Novel stromal cell-derived factor-1 polypeptides, polynucleotides, modulators thereof and methods of use

ABSTRACT

Disclosed herein is a newly identified SDF-1 splice variant molecule, its polypeptide sequence, and the polynucleotides encoding the polypeptide sequence, and active fragments thereof. Also provided is a procedure for producing such polypeptides by recombinant techniques employing, for example, vectors and host cells. Also disclosed are methods for utilizing such polypeptides and modulators thereof for the treatment of diseases, including cancer, immune diseases, infectious diseases, and ischemic diseases.

PRIORITY CLAIM

This application claims the benefit of provisional application60/567,311, filed in the United States Patent and Trademark Office onApr. 30, 2004, the disclosures of which is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to newly identified stromal cell-derivedfactor-1 (“SDF-1”) polypeptides, polynucleotides encoding such,modulators thereof, vectors, host cells, compositions, and kitscontaining such, the methods of making and methods of using suchpolypeptides, polynucleotides, and modulators, such as antibodies, indiagnostic, prophylactic, and therapeutic applications.

BACKGROUND OF THE INVENTION

Chemokines, or chemotactic cytokines, are a class of cytokine moleculescapable of chemotactically attracting migratory cells. Chemokines areessential in attracting cells to inflammatory sites irrespective of theetiology, including immunologic, infective, ischemic, or drug-inducedcauses of inflammation. Chemokines generally are small molecular weightmolecules in the range of about 8-10 kilodaltons (“kD”).

Most chemokines can be divided into three major families, CC, CXC, andCXXXC, based on the number of amino acids (referred to as “X”)separating the two cysteines (referred to as “C”) in the chemokinemolecule. Within the CC and CXC families, chemokines are further groupedinto related sub-families based on amino acid sequence similarity. CCchemokine sub-families include the monocyte chemoattractant protein(“MCP”) sub-family and the sub-families that include macrophageinhibitory protein-1α (“MIP-1α”), macrophage inhibitory protein-1β(“MIP-1β”), and the regulated on activation normal T cell expressed(“RANTES”) subfamily. CXC chemokine sub-families include the IP-10 andMig sub-family, the interleukin-8 (“IL-8”) sub-family, and the PF4sub-family. The chemokines stromal cell-derived factor 1α (“SDF-1α”) andstromal cell-derived factor 1β (“SDF-1β”) form a chemokine family thatis related by amino acid sequence similarity to the both CC and CXCchemokine families.

Chemokines generally exert their effect by binding to chemokinereceptors. CC chemokines typically bind to members of the CCR class ofreceptors, while CXC chemokines typically bind to members of the CXCRclass of receptors. These receptors are involved in regulating theextent and nature of inflammation, and certain receptors tend to belocalized in certain tissues and cells.

Stromal cell-derived factor-1 (“SDF-1”), a member of the CXC chemokinefamily, is a potent chemoattractant for hematopoietic cells, includingbone marrow progenitors (Aiuti et al., J. Exp. Med., 185:111-120(1991)), lymphocytes (Bleul et al., Nature (Lond.), 382:828-833 (1996)),monocytes, and polymorphonuclear cells (Bleul et al., J. Exp. Med.,184:1101-1109 (1996)). SDF-1 also stimulates proliferation of β-cellprogenitors in vitro (Nagasawa et al., Nature (Lond.), 382:635-638(1996)). SDF-1 is thus likely to attract hematopoietic cells inappropriate microenvironments in which they differentiate or proliferatein response to local stimuli. SDF-1 is the ligand for CXCR4, a Gprotein-coupled receptor that is expressed not only in hematopoieticcells but also in a large variety of tissues, such as brain microglia(Lavi et al., Am. J. Pathol., 1035-1042 (1997)), and endothelia (Guptaet al., J. Biol. Chem., 273:4282-4287 (1998)). Accordingly,CXCR4-deficient mice exhibited cardiac defects, abnormal cerebellardevelopment, and anatomical changes of gastrointestinal tractvascularization (Tachibana et al., Nature (Lond.), 393:591-594 (1998)).

SDF-1 is believed to play an important role during embryogenesis andhematopoiesis by recruiting hematopoietic stem cell (“HSC”) precursorsto become bone marrow cells. In the adult, SDF-1 is hypothesized to beinvolved in migration of lymphocytes to lymphoid organs or to sites ofinflammation. SDF-1 is expressed in both splenic red pulp and in lymphnode medullary cords, as well as on high endothelial venules (“HEV”).Moreover, SDF-1 has been found to induce recruitment of an SDF-1responsive cell line to human peripheral lymph nodes grafted into SCIDmice. However, the entire role of SDF-1 in health and disease is not yetclear.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES Brief Description of theFigure

FIG. 1 shows the polypeptide alignment of SEQ. ID. NOS.:9-13. SEQ. ID.NO.:9 is the novel SDF-1 polypeptide of the invention. SEQ. ID.NOS.:10-13 are known sequences from the NCBI database. SEQ. ID.NOS.:14-16 are active fragments of the novel SDF-1 polypeptide,containing exons 2-4, exons 3-4, and exon 4 respectively. SEQ. ID.NO.:22 is the 3′ region from SEQ. ID. NO.:9, which contains the regionnot shown in the public sequence SEQ. ID. NO.:12. SEQ. ID. NO.:23 is the5′ region from SEQ. ID. NO.:9. The alignment was performed using ClustalFormat for T-COFFEE Version_(—)1.37, CPU=0.00 sec, SCORE-76, Nseq=9,Len=140. Table 1 shows the correlation between SEQ. ID NOS. and thesequences as designated in FIG. 1.

BRIEF DESCRIPTION OF THE TABLES

In Table 1, column 1 shows an internal designation identification number(FP ID); column 2 shows the nucleotide sequence identification numberfor the open reading frame of the nucleic acid sequence (SEQ. ID.NO.:N1); column 3 shows the amino acid sequence identification numberfor the polypeptide sequence (SEQ. ID. NO.:P1); column 4 shows thenucleotide sequence identification number for the entire nucleic acidsequence (SEQ. ID. NO.:N0); and column 5 shows the polypeptideidentification number of the source clone or sequence (Source ID).

Table 2 shows the public annotation of the polypeptide sequences of theSequence Listing. Column 1 shows an internal designation identificationnumber of the polypeptide (FP ID); column 2 shows the sourceidentification number of the polypeptide (Source ID); column 3 shows thepredicted length of the polypeptide (Pred Prot Len); column 4 shows thepublic ID number of a best human hit found in the NCBI public databaseNR (Top Human Hit Accession ID); column 5 shows the annotation of the IDnumber set forth in column 4 (Top Human Hit Annotation); column 6comprises the top hit polypeptide length of the sequence set forth incolumn 4 (Top Human Hit % ID); and column 7 shows the length of thematch between the polypeptide and the sequence shown in column 4 TopHuman Match Length).

Table 3 shows information about the polypeptides of the SequenceListing. Column 1 shows an internal designation identification number ofthe polypeptide (FP ID); column 2 shows the source identification numberof the polypeptide (Source ID); column 3 shows the clusteridentification number of the polypeptide (Cluster); column 4 shows theclassification of the polypeptide (Class); column 5 shows the predictedprotein length (Pred Prot Len); column 6 shows an internal parameterwhich designates whether a polypeptide is secreted; treevotes of 0.98and 1 designate that the polypeptide is secreted and treevotes of0.05-0.21 designate that the polypeptide is not secreted (Treevote);column 7 shows the mature protein coordinates (Mature Protein Coords);column 8 shows the signal peptide coordinates (Signal Peptide Coords);column 9 shows an alternate prediction of the mature protein coordinates(Alternate Mature Protein Coords); column 10 shows the number oftransmembrane domains; coordinates of transmembrane domains (TM); andcolumn 11 shows the protein family (Pfam).

Table 4 shows a comparison between the disclosed polypeptide and knownvariants. Column 1 shows an internal designation identification numberof the polypeptide (FP ID); column 2 shows the source identificationnumber of the polypeptide (Source ID); column 3 shows the predictedpolypeptide length of the sequence shown in column 2 (Pred Prot Len);column 4 shows the length of the match between the disclosed polypeptide(FP ID) and the sequence shown in column 2 (Source ID); column 5 showsthe percent identity between the disclosed polypeptide (FP ID) and thesequence shown in column 2 (Source ID); over the length of the disclosedpolypeptide (FP ID); and column 6 shows the percent identity between thedisclosed polypeptide (FP ID) and the sequence shown in column 2 (SourceID) over the length of the sequence shown in column 2 (Source ID).

Table 5 shows the Pfam coordinates of the invention. Column 1 shows aninternal designation identification number of the polypeptide (FP ID);column 2 shows the source identification number of the polypeptide(Source ID); column 3 shows the name of the Pfam domain; and column 4shows the start and stop coordinates of the pfam domain within thepolypeptide.

INDUSTRIAL APPLICABILITY

The polypeptide and modulator compositions and methods of the inventionare useful in the diagnosis, treatment, and/or prevention ofproliferative diseases, inflammatory and immune or autoimmune diseases,hematopoeitic diseases, infectious diseases, ischemic diseases, andmetabolic diseases. They are also useful in modulating an immuneresponse and inhibiting tumor growth.

DISCLOSURE OF THE INVENTION Summary of the Invention

The inventors herein have found novel SDF-1 polypeptides,polynucleotides encoding such, as well as modulators thereof that areuseful in stimulating and inhibiting certain biological activities. Thepolypeptides of the present invention may be used for diseases relatingto insufficient or abnormal proliferation of hematopoietic cells,neuronal enhancement or depression, and immunological enhancement anddepression. For example, SDF-1 polypeptides, polynucleotides, andmodulators thereof can be used for treatment or prevention ofinflammatory diseases including rheumatoid arthritis and ulcerativecolitis, hematopoietic stem cytopenia after bone marrow transplantation,leukocytopenia, thrombocytopenia, B lymphopenia and T lymphopenia afterchemotherapy, anemia, infectious diseases, cancer, leukocytosis, HIVinfection, neurodegenerative diseases including Alzheimer's disease andmultiple sclerosis, neuronal injury, bone disorders such asosteoporosis, and/or tissue repair.

The present invention provides newly identified SDF-1 variantpolypeptides, as well as isolated polynucleotides encoding thepolypeptides and expression vectors containing the isolatedpolynucleotides. Accordingly, the invention provides methods andcompositions for treatment, prevention, and diagnosis of diseases orconditions associated with the polypeptides of the invention, as well asthe polynucleotides encoding the polypeptides.

The invention also provides a method for producing the disclosedpolypeptides by cell free expression and culturing host cellstransformed with a recombinant expression vector that contains thepolypeptides encoding nucleic acids under conditions appropriate forexpression of polypeptides, and recovering the expressed polypeptidesfrom the culture.

The invention further provides modulators of the polypeptides of theinvention, including but not limited to antibodies thereto, fortreatment, prevention, and diagnosis of diseases or conditionsassociated with their respective receptors. Antibodies of the inventionmay specifically bind to or interfere with the activity of polypeptidesof the invention, wherein such polypeptides contain at least a sequenceof six contiguous amino acid residues chosen from the polypeptides ofthe Sequence Listing.

The invention yet further identifies further uses for the polypeptidesof the invention, as well as the isolated polynucleotides encoding thepolypeptides and modulators thereto.

In another aspect, the invention provides compositions containing thepolypeptides of the invention, and a vehicle such as pharmaceuticallyacceptable carrier or excipient, wherein the compositions are useful fortreatment or prophylaxis of diseases in animals.

The invention also provides a method for stimulating an immune responsein a subject with the polypeptides of the invention and the isolatedpolynucleotides encoding the polypeptides of the invention.

The invention further provides a method for treating or preventing aninfection in a subject by use of the polypeptides of the invention, aswell as the isolated polynucleotides encoding the polypeptides.

The invention yet further provides a method for modulating an immuneresponse in a subject by use of an agonist or an antagonist of thepolypeptides of the invention.

The invention provides a method of treating diseases, such asinflammatory diseases, autoimmune diseases, ischemia related disorders,such as stroke, myocardial infarction, and fulminant liver failure,cancer, and infectious diseases.

The invention identifies nucleotide and polypeptide targets fordiagnosis and therapeutic intervention of the disease states describedherein, and provides methods for diagnosis and treating these diseasesby intervening with these targets. The invention provides the nucleicacid and amino acid sequences of these targets in the Sequence Listing.

The invention provides a first isolated nucleic acid molecule comprisinga first polynucleotide or amin acid sequence chosen from chosen from theSequence Listing; biologically active fragments thereof, and acomplement thereof. This first isolated nucleic acid molecule can bechosen from a cDNA molecule, a genomic DNA molecule, a cRNA molecule, asiRNA molecule, a RNAi molecule, an mRNA molecule, an antisensemolecule, and a ribozyme. The invention also provides a double-strandedisolated nucleic acid molecule comprising the first nucleic acidmolecule and its complement.

The invention also provides a second isolated nucleic acid moleculecomprising a second polynucleotide sequence that hybridizes to a firstpolynucleotide sequence comprising a nucleotide sequence chosen from theSequence Listing under high stringency conditions.

The invention further provides an isolated polypeptide comprising anamino acid sequence chosen from chosen from the Sequence Listing. Itprovides an isolated polypeptide encoded by a first nucleic acidmolecule as described above.

The invention yet further provides vectors and host cells. It provides avector comprising a first nucleic acid molecule as described above and apromoter that regulates the expression of the nucleic acid molecule.This promoter can be chosen from one that is naturally contiguous to thenucleic acid molecule and one that is not naturally contiguous to thenucleic acid molecule. It can be an inducible promoter, aconditionally-active promoter (such as the cre-lox promoter), aconstitutive promoter, and/or a tissue-specific promoter. Host cells ofthe invention include recombinant host cells comprising a first nucleicacid molecule as described above, an isolated polypeptide as describedabove, and the vectors as described above. Host cells of the inventioncan be prokaryotic cells or eukaryotic cells. Eukaryotic host cells ofthe invention include human cells, non-human mammalian cells, insectcells, fish cells, plant cells, and fungal cells.

The invention provides a non-human animal injected with the firstnucleic acid molecule described above. The animal may be geneticallymodified with this first nucleic acid molecule. It may be injected witha polypeptide of the invention, as described above.

In another aspect, the invention provides a nucleic acid compositioncomprising the first nucleic acid molecule described above and acarrier. This carrier may be a pharmaceutically acceptable carrier or anexcipient, which may, in turn, be chosen from saline, phosphate bufferedsaline, and a lipid based formulation. It provides a polypeptidecomposition comprising a polypeptide as described above and a carrier,which may also be a pharmaceutically acceptable carrier or an excipient.It also provides a vector composition comprising the vector as describedabove and a carrier; the carrier may also be a pharmaceuticallyacceptable carrier or an excipient. It further provides a host cellcomposition comprising the host cell as described above and a carrier;the carrier may also be a pharmaceutically acceptable carrier or anexcipient.

In yet another aspect, the invention provides a method of producing arecombinant host cell by providing a composition comprising a vectorthat comprises the first nucleic acid molecule as described above andallowing a host cell to come into contact with the vector to form arecombinant host cell. The invention provides a method of producing apolypeptide by providing a composition comprising a recombinant hostcell as described above and culturing the recombinant host cell toproduce the polypeptide. It also provides a method of producing apolypeptide by providing the first nucleic acid as described above andexpressing the nucleic acid molecule in a cell free expression system toproduce the polypeptide. The cell free expression system can be chosenfrom a wheat germ lysate expression system, a rabbit reticulocyteexpression system, and an E. coli lysate expression system.

In yet a further aspect the invention provides a diagnostic kitcomprising a composition comprising a first polynucleotide molecule asdescribed above and a vehicle. It also provides a diagnostic kitcomprising an antibody that specifically binds to a polypeptide of theinvention as described above or a biologically active fragment thereof.It further provides a diagnostic kit comprising a polypeptide of theinvention as described above or a biologically active fragment thereof.

The invention provides a method of determining presence of a firstpolynucleotide molecule as described above by providing this nucleicacid molecule, allowing it to interact with the sample; and determiningwhether specific binding has occurred. It provides a method ofdetermining the presence of an antibody specific to a polypeptide of theinvention as described above or a biologically active fragment thereofby providing a composition comprising the polypeptide, allowing thepolypeptide to interact with the sample, and determining whetherspecific binding has occurred between the polypeptide and the antibody.

The invention provides an antibody that specifically binds to orinterferes with activity of a polypeptide of the invention as describedabove or a biologically active fragment thereof. This antibody may be apolyclonal antibody, a monoclonal antibody, a single chain antibody, andan active fragment of any of these. This antibody may be a fragment,including an antigen binding fragment, an Fc fragment, a cdr fragment, aframework fragment, a variable region of an immunoglobulin, a constantregion of an immunoglobulin, and/or a combination thereof. The inventionalso provides an aptamer that specifically binds to or interferes withthe activity of a polypeptide of the invention as described above or abiologically active fragment thereof.

The invention provides a method of treatment of a B-cell deficiency in asubject by providing a composition containing a polypeptide chosen fromchosen from the Sequence Listing, or an active fragment thereof, and acarrier; and administering the composition to a subject. Thiscomposition may further comprise a soluble factor. Suitable solublefactors for use in the invention include interleukins, for example, IL-7and cytokines. This method can be used to treat Brutonagammaglobulinemia. It can be performed by administering the compositionto a subject either locally or systemically. The carrier may be apharmaceutically acceptable carrier or an excipient.

The invention also provides a method of treatment of a plateletdeficiency in a subject by providing a composition containing apolypeptide chosen from chosen from the Sequence Listing, or an activefragment thereof, and a carrier; and administering the composition to asubject. This method can be used to treat thrombocytopenia. It can beperformed by administering the composition to a subject either locallyor systemically. The carrier may be a pharmaceutically acceptablecarrier or an excipient.

The invention further provides a method of stimulating lymphocyte growthor proliferation in a subject by providing a composition containing thepolypeptide chosen from chosen from the Sequence Listing, or an activefragment thereof, and a carrier; and administering the composition to asubject. It can be performed by administering the composition to asubject either locally or systemically. The carrier may be apharmaceutically acceptable carrier or an excipient. It can be performedby administering the composition after stem cell transplant.

The invention yet further provides a method for treating an adverseeffect of a cancer therapy in a subject by administering a compositioncontaining the polypeptide chosen from chosen from the Sequence Listing,or an active fragment thereof, and a carrier, collecting a population ofstem cells from the subject, and administering the population of stemcells to the subject. The cancer therapy may be body irradiation, abone-marrow depleting agent, for example a cytotoxic bone-marrowdepleting agent. The method may be performed by administering the stemcells prior to, substantially contemporaneously with, or after thecancer therapy.

The invention provides a method of treating diabetes in a subject byproviding a composition containing the polypeptide chosen from chosenfrom the Sequence Listing, or an active fragment thereof, and a carrierand administering the composition to the subject. It can be performed byadministering the composition to a subject either locally orsystemically. The carrier may be a pharmaceutically acceptable carrieror an excipient.

The invention also provides a method of promoting angiogenesis in asubject by providing a composition containing the polypeptide chosenfrom chosen from the Sequence Listing or an active fragment thereof, anda carrier; and the polypeptide chosen from chosen from the SequenceListing, or an active fragment thereof, and a carrier and administeringthe composition to the subject. It can be performed by administering thecomposition to a subject either locally or systemically. The carrier maybe a pharmaceutically acceptable carrier or an excipient.

The invention further provides a method of modulating an immune responsein a subject by providing a modulator of a polypeptide chosen from anyof SEQ. ID. NOS.:9 and 14-16; or an active fragment thereof, andadministering the composition to the subject. The modulator may be anantibody, which may, for example, be a monoclonal antibody, a polyclonalantibody, a cdr fragment, a framework fragment, a single chain antibody,and an active fragment of an antibody. This method can be performed tomodulate the suppression of inflammation and/or autoimmune diseases.This method can be performed to modulate the immune response by treatingrheumatoid arthritis, osteoarthritis, psoriasis, inflammatory boweldisease, multiple sclerosis, systemic lupus erythematosus (SLE), Graves'disease, immunoproliferative disease lymphadenopathy (IPL),angioimmunoproliferative lymphadenopathy (AIL), and/or immunoblastivelymphadenopathy (IBL). This modulation can be performed by administeringthe composition to the subject locally or systemically in apharmaceutically acceptable carrier or an excipient.

The invention yet further provides a method for treating or preventingan infection in a subject by providing a composition containing thepolypeptide chosen from SEQ. ID. NOS.:5-6, 19, 26-27, or an activefragment thereof, and a carrier; and administering the composition to asubject. This method can be used to treat or prevent a bacterialinfection, a mycoplasma infection, a fungal infection, and/or a viralinfection, for example, human immunodeficiency virus (HIV). It can beperformed by administering the composition to a subject either locallyor systemically. The carrier may be a pharmaceutically acceptablecarrier or an excipient.

The invention provides a method for treating or preventing an ischemicdisease, including but not limited to stroke, myocardial infarction, andfulminant liver failure, in a subject by providing a compositioncontaining a polynucleotide or polypeptide chosen from the SequenceListing or an active fragment thereof, and a carrier. It can beperformed by administering the composition to a subject either locallyor systemically. The carrier may be a pharmaceutically acceptablecarrier or an excipient.

The invention also provides a method for inhibiting tumor growth in asubject by providing a composition containing a polynucleotide orpolypeptide chosen from the Sequence Listing or an active fragmentthereof, and a carrier. It can be performed by administering thecomposition to a subject either locally or systemically. The carrier maybe a pharmaceutically acceptable carrier or an excipient. This methodcan be performed to inhibit tumor growth, wherein the tumor is comprisedof solid tumor cells or leukemic cells.

The invention further provides a method of treating a cancer in asubject by providing a composition containing a polynucleotide orpolypeptide chosen from the Sequence Listing or an active fragmentthereof, and a carrier. It can be performed by administering thecomposition to a subject either locally or systemically. The carrier maybe a pharmaceutically acceptable carrier or an excipient. This methodcan be performed to treat cancer, wherein the cancer is a solid tumor ora leukemia.

The invention yet further provides a method of treating a cancer in asubject by providing a composition containing a polynucleotide orpolypeptide chosen from the Sequence Listing or an active fragmentthereof, and a carrier. It can be performed by administering thecomposition to a subject either locally or systemically. The carrier maybe a pharmaceutically acceptable carrier or an excipient. This methodcan be performed to treat an allergy, wherein the allergy is asthma.

In another aspect, the invention provides for the use of thepolynucleotides or polypeptides of the Sequence Listing or an activefragment thereof as a target for screening for a modulator. Targets caninclude a small molecule drug, an antibody, and an aptamer.

The invention provides a method of modulating an immune condition in asubject by providing a modulator of a polypeptide chosen from theSequence Listing and active fragments thereof; and administering themodulator to the subject. The modulator can be an antibody and theantibody can be a monoclonal antibody, a polyclonal antibody, a cdrfragment, a framework fragment, a single chain antibody, and/or anactive fragment of an antibody. The immune condition may compriseinflammation and the modulation may comprise suppression. The immunecondition may comprise autoimmune disease and the modulation maycomprise suppression. The immune condition may comprise rheumatoidarthritis, osteoarthritis, psoriasis, inflammatory bowel disease,multiple sclerosis, myocardial infarction, stroke, and/or fulminantliver failure. The modulation may comprise suppression.

The invention also provides a method of enhancing immune response to avaccine in a subject by providing a polypeptide composition comprising asubstantially purified polypeptide chosen from the Sequence Listing andactive fragments thereof; providing a vaccine composition; andadministering the polypeptide composition and the vaccine composition tothe subject. The polypeptide composition may be administered to thesubject prior to, substantially contemporaneously with, or afteradministering the vaccine composition.

The invention provides a method for promoting tissue regeneration in asubject by providing a nucleic acid molecule comprising a polynucleotideor polypeptide sequence chosen from chosen from the Sequence Listing; apolynucleotide sequence encoding a nucleotide chosen from chosen fromthe Sequence Listing; biologically active fragments of these, and/orcomplements of these and administering the nucleic acid to the subject.The method can be performed by administering the molecule locally, forexample, into a tissue. Suitable tissues include brain, heart, pancreas,lung, liver, and bone. The method can be performed wherein the moleculeis present in a cell, for example, a heart cell, a brain cell, apancreatic cell, a lung cell, a liver cell, or a bone cell. The heartcell may, for example, be a cardiac fibroblast.

The invention also provides a method for promoting tissue regenerationin a subject by providing a polypeptide comprising an amino acidsequence chosen from chosen from the Sequence Listing; biologicallyactive fragments of these, and complements of these; and administeringthe amino acid sequence to the subject. The method can be performed byadministering the molecule locally, for example, into a tissue. Suitabletissues include brain, heart, pancreas, lung, liver and bone. The methodcan be performed wherein the molecule is present in a cell, for example,a heart cell, a brain cell, a pancreatic cell, a lung cell, a livercell, or a bone cell. This method can be performed by administering oneor more stem cells to the subject prior to, substantiallycontemporaneously with, or after administering the nucleic acid moleculeor polypeptide. In an embodiment, the method is performed bytransfecting the cells of a subject with SDF-1, providing them to anorgan of the subject, and providing the subject with stem cells, forexample, mesenchymal stem cells, either before, during, or afterproviding the transfected cells. In an embodiment, the method isperformed by transfecting the cardiac cells of a subject with SDF-1,providing them to the heart, for example by injection, and providing thesubject with stem cells, for example, mesenchymal stem cells, eitherbefore, during, or after the transfected cardiac cells.

The invention provides a cell transfected with an isolated nucleic acidmolecule comprising a first polynucleotide sequence chosen from SEQ. ID.NOS.: 1, 6-8, and 17; a polynucleotide sequence encoding a polypeptideof SEQ. ID. NO.:9, 14-16, 22-23; biologically active fragments thereof,and a complement thereof. This cell may be a heart cell, a brain cell, apancreatic cell, a lung cell, a liver cell, a skin cell, a bone cell, amesenchymal stem cell, a progenitor cell, an adult stem cell, or anembryonic stem cell.

The invention provides a method of treating a subject who can benefitfrom receiving such a polynucleotide, or an isolated polypeptidecomprising an amino acid sequence, wherein the amino acid sequence ischosen from SEQ. ID. NO.:9, 14-16, and 22-23 by providing a compositioncomprising such a polynucleotide or polypeptide and administering thecomposition to the subject. The subject may be treated for acardiovascular disease, brain disease, cancer, infection, diabetes, bonedisease, lung disease, liver disease, skin disease, burn, stroke,trauma, injury, or deficiency. The composition may further comprise apharmaceutically acceptable carrier or excipient. It may be administeredlocally or systemically. The composition may be administered incombination with another therapeutic, wherein the other therapeutic, forexample, a growth factor or cytokine, is administered before, after, orsubstantially contemporaneously with the composition. The composition isadministered at a therapeutically effective dose.

Suitable growth factors include fibroblast growth factor, plateletderived growth factor, insulin-like growth factor, epidermal growthfactor, GCSF, GM-CSF, TGF-α, TGF-β, PD-ECGF, pre-B cell enhancing factor(PBEF), an osteogenic factor, c-kit ligand, stem cell factor, TNF-α, andmuteins or variants of any of these which have growth factor activity.The fibroblast growth factor may be chosen from FGF2, FGF4, FGF8, FGF9,and muteins or variants of any of these which have growth factoractivity. Suitable platelet derived growth factors include PDGF-AA,PDGF-BB, PDGF-AB, and muteins or variants of any of these which havegrowth factor activity. Suitable cytokines include IL-13.

The invention provides a polypeptide comprising the amino acid sequenceof amino acids 22-88 of CLN00235738_(—)5pv1.a and further comprising atleast one additional amino acid chosen from amino acids 89-140, whereinpolypeptide comprises a contiguous sequence of amino acids chosen fromCLN00235738_(—)5pv1.a as shown in FIG. 1.

DEFINITIONS

The terms used herein have their ordinary meanings, as set forth below,and can be further understood in the context of the specification.

The terms “polynucleotide,” “nucleotide,” “nucleic acid,” “nucleic acidmolecule,” “nucleic acid sequence,” “polynucleotide sequence,” and“nucleotide sequence” are used interchangeably herein to refer topolymeric forms, both double- and single-stranded, of nucleotides of anylength. The polynucleotides can contain deoxyribonucleotides,ribonucleotides, and/or their analogs or derivatives.

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include naturally-occurring amino acids, coded and non-coded aminoacids, chemically or biochemically modified, derivatized, or designeramino acids, amino acid analogs, peptidomimetics, and depsipeptides, andpolypeptides having modified, cyclic, bicyclic, depsicyclic, ordepsibicyclic peptide backbones. The term includes single chain proteinas well as multimers. The term also includes conjugated proteins, fusionproteins, including, but not limited to, glutathione S-transferase (GST)fusion proteins, fusion proteins with a heterologous amino acidsequence, fusion proteins with heterologous and homologous leadersequences, fusion proteins with or without N-terminal methionineresidues, pegolyated proteins, and immunologically tagged, or his-taggedproteins. The term also includes peptide aptamers.

An “isolated,” “purified,” “substantially isolated,” or “substantiallypurified” molecule (such as a polypeptide or polynucleotide) is one thathas been manipulated to exist in a higher concentration than in nature.For example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically. As usedherein, an “isolated,” “purified,” “substantially isolated,” or“substantially purified” molecule includes recombinant molecules.

By “fragment” is intended a polynucleotide or polypeptide consisting ofonly a part of the intact full-length or naturally occurringpolynucleotide or polypeptide sequence and structure. A polypeptidefragment can include e.g., a C-terminal deletion, an N-terminaldeletion, and/or an internal deletion of a native polypeptide or anextracellular domain of a transmembrane protein. A fragment of a proteinwill generally include at least about 5-10, 15-25, or 20-50 or morecontiguous amino acid residues of the full-length molecule, at leastabout 15-25 contiguous amino acid residues of the full-length molecule,or any integer between 5 amino acids and the full-length sequence.

A “complement” of a nucleic acid molecule is a one that is comprised ofits complementary base pairs. Deoxyribonucleotides with the base adenineare complementary to those with the base thymidine, anddeoxyribonucleotides with the base thymidine are complementary to thosewith the base adenine. Deoxyribonucleotides with the base cytosine arecomplementary to those with the base guanine, and deoxyribonucleotideswith the base guanine are complementary to those with the base cytosine.Ribonucleotides with the base adenine are complementary to those withthe base uracil, and deoxyribonucleotides with the base uracil arecomplementary to those with the base adenine. Ribonucleotides with thebase cytosine are complementary to those with the base guanine, anddeoxyribonucleotides with the base guanine are complementary to thosewith the base cytosine.

A “promoter,” as used herein, is a DNA regulatory region capable ofbinding RNA polymerase in a mammalian cell and initiating transcriptionof a downstream (3′ direction) coding sequence operably linked thereto.For purposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes. Promoters include those that are naturallycontiguous to a nucleic acid molecule and those that are not naturallycontiguous to a nucleic acid molecule. Additionally, a promoter includesinducible promoters, conditionally active promoters, such as a cre-loxpromoter, constitutive promoters, and tissue specific promoters.

A “vector” is a plasmid that can be used to transfer DNA sequences fromone organism to another or to express a gene of interest.

“Expression of a nucleic acid molecule” refers to the conversion of theinformation contained in the molecule, into a gene product. A geneproduct can be the direct transcriptional product of a gene (e.g., mRNA,tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other typeof RNA) or a peptide or polypeptide produced by translation of an mRNA.Gene products also include RNAs which are modified, by processes such ascapping, polyadenylation, methylation, and editing, and proteinsmodified by, for example, methylation, acetylation, phosphorylation,ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

The term “host cell” includes an individual cell, cell line, cellculture, or cell in vivo, which can be or has been a recipient of anypolynucleotides or polypeptides of the invention, for example, arecombinant vector, an isolated polynucleotide, an antibody or a fusionprotein. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology,physiology, or in total DNA, RNA, or polypeptide complement) to theoriginal parent cell due to natural, accidental, or deliberate mutationand/or change. Host cells can be prokaryotic or eukaryotic, includingmammalian, insect, amphibian, reptilian, crustacean, avian, fish, plant,and fungal cells. A host cell includes cells transformed, transfected,transduced, or infected in vivo or in vitro with a polynucleotide of theinvention, for example, a recombinant vector. A host cell whichcomprises a recombinant vector of the invention may be called a“recombinant host cell.”

The term “recombinant” as used with respect to a host cell means a hostcell into which a recombinant polynucleotide has been introduced.

A “biologically active” entity, or an entity having “biologicalactivity,” is one or more entity having structural, regulatory, orbiochemical functions of a naturally occurring molecule or any functionrelated to or associated with a metabolic or physiological process.Biologically active polynucleotide fragments are those exhibitingactivity similar, but not necessarily identical, to an activity of apolynucleotide of the present invention. The biological activity caninclude an improved desired activity, or a decreased undesirableactivity. For example, an entity demonstrates biological activity whenit participates in a molecular interaction with another molecule, suchas hybridization, when it has therapeutic value in alleviating a diseasecondition, when it has prophylactic value in inducing an immuneresponse, when it has diagnostic value in determining the presence of amolecule, such as a biologically active fragment of a polynucleotidethat can, for example, be detected as unique for the polynucleotidemolecule, or that can be used as a primer in a polymerase chainreaction. A biologically active polypeptide or fragment thereof includesone that can participate in a biological reaction, for example, one thatcan serve as an epitope or immunogen to stimulate an immune response,such as production of antibodies, or that can participate in stimulatingor inhibiting signal transduction by binding to ligands receptors orother proteins, or nucleic acids; or activating enzymes or substrates.

The terms “antibody” and “immunoglobulin” refer to a protein, forexample, one generated by the immune system, synthetically, orrecombinantly, that is capable of recognizing and binding to a specificantigen; antibodies are commonly known in the art. Antibodies mayrecognize polypeptide or polynucleotide antigens. The term includesactive fragments, including for example, an antigen binding fragment ofan immunoglobulin, a variable and/or constant region of a heavy chain, avariable and/or constant region of a light chain, a complementaritydetermining region (cdr), and a framework region. The terms includepolyclonal and monoclonal antibody preparations, as well as preparationsincluding hybrid antibodies, altered antibodies, chimeric antibodies,hybrid antibody molecules, F(ab′)₂ and F(ab) fragments; Fv molecules(for example, noncovalent heterodimers), dimeric and trimeric antibodyfragment constructs; minibodies, humanized antibody molecules, and anyfunctional fragments obtained from such molecules, wherein suchfragments retain specific binding.

The term “specific binding,” in the context of antibody binding, refersto high avidity and/or high affinity binding of an antibody to aspecific epitope. Hence, an antibody that binds specifically to oneepitope (a “first epitope”) and not to another (a “second epitope”) is a“specific antibody.” An antibody specific to a first epitope may crossreact with and bind to a second epitope if the two epitopes sharehomology or other similarity.

The term “specific binding,” in the context of a polynucleotide, refersto hybridization under stringent conditions. Conditions that increasestringency of both DNA/DNA and DNA/RNA hybridization reactions arewidely known and published in the art. See, for example, Sambrook,Sambrook et al. Molecular Cloning, A Laboratory Manual, 2000.

“Subject,” “individual,” “host,” and “patient” are used interchangeablyherein to refer to mammals, including, but not limited to, rodents,simians, humans, felines, canines, equines, bovines, porcines, ovines,caprines, mammalian laboratory animals, mammalian farm animals,mammalian sport animals, and mammalian pets.

A “patient sample” is any biological specimen derived from a patient;the term includes, but is not limited to, biological fluids such asblood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph,dialysis fluid, lavage fluid, semen, and other liquid samples, as wellas cell and tissues of biological origin. The term also includes cellsor cells derived therefrom and the progeny thereof, including cells inculture, cell supernatants, and cell lysates. It further includes organor tissue culture-derived fluids, tissue biopsy samples, tumor biopsysamples, stool samples, and fluids extracted from physiological tissues,as well as cells dissociated from solid tissues, tissue sections, andcell lysates. This definition encompasses samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents, solubilization, or enrichment for certain components,such as polynucleotides or polypeptides. Also included in the term arederivatives and fractions of patient samples. A patient sample may beused in a diagnostic, prognostic, or other monitoring assay.

The term “modulate” refers to the production, either directly orindirectly, of an increase or a decrease, a stimulation, inhibition,interference, or blockage in a measured activity when compared to asuitable control. A “modulator” of a polypeptide or polynucleotide or an“agent” are terms used interchangeably herein to refer to a substancethat affects, for example, increases, decreases, stimulates, inhibits,interferes with, or blocks a measured activity of the polypeptide orpolynucleotide, when compared to a suitable control. An agent whichmodulates a biological activity of a subject polypeptide orpolynucleotide increases or decreases the activity or binding at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 50%, at least about 80%, or at least about 2-fold, atleast about 5-fold, or at least about 10-fold or more when compared to asuitable control.

“Treatment,” as used herein, covers any administration or application ofremedies for disease in a mammal, including a human, and includesinhibiting the disease, i.e., arresting its development, or relievingthe disease, i.e., causing regression, or restoring or repairing a lost,missing, or defective function; or stimulating an inefficient process.In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells or cancer cells, preventing replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease. In thecontext of an autoimmune disease, the term “treating” includes any orall of: preventing replication of cells associated with an autoimmunedisease state including, but not limited to, cells capable of producingan autoimmune antibody, lessening the autoimmune-antibody burden andameliorating one or more symptoms of an autoimmune disease. In thecontext of an infectious disease, the term “treating” includes any orall of preventing the growth, multiplication or replication of thepathogen that causes the infectious disease and ameliorating one or moresymptoms of an infectious disease. In the context of an ischemicdisease, the term “treating” includes any or all of preventing thegrowth, multiplication or replication of the pathogen that causes theischemic disease and ameliorating one or more symptoms of an ischemicdisease.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Treatment and prophylaxis can be administered to anorganism, including a human, or to a cell in vivo, in vitro, or ex vivo,and the cell subsequently administered to the subject.

“Disease” refers to any condition, infection, disorder, or syndrome thatrequires medical intervention or for which medical intervention isdesirable. Such medical intervention can include treatment, diagnosis,and/or prevention.

“Cancer” is any abnormal cell or tissue growth, for example, a tumor,whether malignant, pre-malignant, or non-malignant. It is characterizedby uncontrolled proliferation of cells that may or may not invade thesurrounding tissue and, hence, may or may not metastasize to new bodysites. Cancer encompasses carcinomas, which are cancers of epithelialcells; carcinomas include squamous cell carcinomas, adenocarcinomas,melanomas, and hepatomas. Cancer also encompasses sarcomas, which aretumors of mesenchymal origin; sarcomas include osteogenic sarcomas,leukemias, and lymphomas. Cancers may involve one or more neoplasticcell type.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material, formulationauxiliary, or excipient of any conventional type. A pharmaceuticallyacceptable carrier is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation.

A “composition” herein refers to a composition that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses, or powders.

Nucleic Acids and Polypeptides

The present invention provides nucleic acid molecules containing apolynucleotide encoding a newly identified SDF-1 variant polypeptidehaving the amino acid sequences as shown in the Sequence Listing (SEQ.ID. NO.:9). The isolated SDF-1 variants of the invention were identifiedby bioinformatic analysis of multiple SDF-1 clones.

Fragments of the full length SDF-1 and SDF-1 variants may be used ashybridization probes for cDNA libraries to isolate the full length geneand to isolate other genes which have a high sequence similarity or asimilar biological activity. Probes of this type can have at least 30bases and may comprise, for example, 50 or more bases. The probe mayalso be used in a screening procedure to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain complete SDF-1 genes, including regulatory and promoterregions, exons, and introns. An example of such a screen would includeisolating the coding regions of SDF-1 genes by using a known nucleicacid sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to a gene of thepresent invention can be used to screen a human cDNA, a genomic DNA, ora mRNA library to identify complementary library components.

The present invention further relates to polynucleotides which hybridizeto the described sequences if there is at least 91%, at least 92%, or atleast 95% identity between the sequences. The present invention relatesto polynucleotides which hybridize under stringent conditions to thedescribed polynucleotides. Stringent conditions generally includecondition under which hybridization will occur only if there is at least95%, or at least 97% identity between the sequences. For example,overnight incubation at 42° C. in a solution containing 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C., constitute stringent conditions. Thepolynucleotides which hybridize to the hereinabove describedpolynucleotides encode polypeptides which may retain substantially thesame biological function or activity as the mature polypeptide.

Alternatively, the polynucleotide may have at least 20 bases, at least30 bases, or at least 50 bases which hybridize to a polynucleotide ofthe present invention and which has an identity thereto, as hereinabovedescribed, and which may or may not retain activity.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, at least a 90% identity, or at least a 95%identity to a polynucleotide which encodes the polypeptides set forth inthe Sequence Listing, as well as fragments thereof, which fragments haveat least 30 bases or at least 50 bases, and to polypeptides encoded bysuch polynucleotides.

Using the information provided herein, such as the nucleotide sequencesset forth in the Sequence Listing, nucleic acid molecules of the presentinvention encoding a SDF-1 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Nucleic acids of the invention are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Polypeptides and antibodiesdirected to those polypeptides are useful for providing immunologicalprobes for the differential identification of tissues or cell types.

Polypeptides and Fragments

The invention further provides an isolated SDF-1 and SDF-1 polypeptidecontaining the amino acid sequences encoded by the nucleotide sequencesset forth in the Sequence Listing, the amino acid sequences set forth inthe Sequence Listing, or a peptide or polypeptide comprising a fragmentof such a polypeptide.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs, orderivatives of the SDF-1 molecules. Variants may occur naturally, suchas a natural allelic variant, i.e., one of several alternate forms of agene occupying a given chromosomal locus Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985)). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions, or additions. The substitutions, deletions, or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. These may take the form of silent substitutions,additions, or deletions which do not alter the properties or activitiesof the described SDF-1 proteins, or portions thereof.

In an embodiment, the invention provides nucleic acid molecules encodingmature proteins, i.e., those with cleaved signal peptide or leadersequences, e.g., as shown in the Sequence Listing. Further embodimentsinclude an isolated nucleic acid molecule comprising a polynucleotidehaving a nucleotide sequence at least 93% identical, or at least 95%,96%, 97%, 98%, or 99% identical to a polynucleotide from the SequenceListing, a polypeptide encoded by a polynucleotide shown in the SequenceListing, a polypeptide shown in the Sequence Listing, or a biologicallyactive fragment of any of these.

A polynucleotide having a nucleotide sequence at least, for example, 95%identical to a reference nucleotide sequence encoding an SDF-1polypeptide is one in which the nucleotide sequence is identical to thereference sequence except that it may include up to five point mutationsper each 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 93%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, thenucleotide sequences set forth in the Sequence Listing can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, Madison, Wis.). Bestfit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981), to find the best segment of homology between twosequences. When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, of course, such that the percentage of identity iscalculated over the full length of the reference nucleotide sequence andthat gaps in homology of up to 5% of the total number of nucleotides inthe reference sequence are allowed.

The present application is directed to nucleic acid molecules at least93%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequencesset forth in the Sequence Listing irrespective of whether they encode apolypeptide having SDF-1 activity. Even where a particular nucleic acidmolecule does not encode a polypeptide having SDF-1 activity, one ofskill in the art would know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having SDF-1 activity include, inter alia,(1) isolating the SDF-1 gene or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide the precise chromosomal location of theSDF-1 genes, as described in Verna et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and Northern blotanalysis for detecting SDF-1 mRNA expression in specific tissues.

The present application is also directed to nucleic acid moleculeshaving sequences at least 93%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of the Sequence Listing which, encode apolypeptide having SDF-1 polypeptide activity, i.e., a polypeptideexhibiting activity either identical to or similar, but not identical,to an activity of the SDF-1 polypeptides of the invention, as measuredin a particular biological assay. For example, the SDF-1 polypeptides ofthe present invention may either stimulate or inhibit the proliferationof various mammalian cells, as demonstrated below.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 93%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the nucleic acidsequences set forth in the Sequence Listing will encode a polypeptidehaving SDF-1 polypeptide activity. In fact, since multiple degeneratevariants of these nucleotide sequences encode the same polypeptide, thiswill be clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat a reasonable number of nucleic acid molecules that are notdegenerate variants will also encode a polypeptide having SDF-1polypeptide activity, the skilled artisan is fully aware of amino acidsubstitutions that are either less likely or not likely to significantlyaffect protein function (e.g., replacing one aliphatic amino acid with asecond aliphatic amino acid), as further described below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatednucleic acid molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof SDF-1 polypeptides or fragments thereof by recombinant techniques.The vector may be, for example, a phage, plasmid, viral, or retroviralvector. Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The present invention provides recombinant vectors that contain, forexample, nucleic acid constructs that encode secretory leader sequencesand a selected heterologous polypeptide of interest, and host cells thatare genetically engineered with the recombinant vectors. Selectedheterologous polypeptides of interest in the present invention include,for example, an extracellular fragment of a secreted protein, a type Imembrane protein, a type II membrane protein, a multi-membrane protein,and a soluble receptor. These vectors and host cells can be used for theproduction of polypeptides described herein, including fragments thereofby conventional recombinant techniques. The vector may be, for example,a phage, plasmid, viral or retroviral vector. Retroviral vectors may bereplication competent or replication defective. As above, in the lattercase, viral propagation generally will occur only in complementing hostcells.

The polynucleotides may be joined to a vector containing a secretoryleader sequence and a selectable marker for propagation in a host.Generally, a plasmid vector is introduced in a precipitate, such as acalcium phosphate precipitate, or in a complex with a charged lipid. Ifthe vector is a virus, it may be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert can be operatively linked to an appropriate promoter,such as the phage lambda PL promoter; the E. coli lac, trp, phoA and tacpromoters; the SV40 early and late promoters; and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs can include a translationinitiating codon at the beginning and a termination codon (UAA, UGA, orUAG) appropriately positioned at the end of the polypeptide to betranslated.

As indicated, the expression vectors may include at least one selectablemarker. Such markers include dihydrofolate reductase, G418 or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

The selectable markers are genes that confer a phenotype on a cellexpressing the marker, so that the cell can be identified underappropriate conditions. Generally, a selectable marker allows theselection of transformed cells based on their ability to thrive in thepresence or absence of a chemical or other agent that inhibits anessential cell function. Suitable markers, therefore, include genescoding for proteins which confer drug resistance or sensitivity thereto,impart color to, or change the antigenic characteristics of those cellstransfected with a molecule encoding the selectable marker, when thecells are grown in an appropriate selective medium. For example,selectable markers include cytotoxic markers and drug resistancemarkers, whereby cells are selected by their ability to grow on mediacontaining one or more of the cytotoxins or drugs; auxotrophic markersby which cells are selected for their ability to grow on defined mediawith or without particular nutrients or supplements, such as thymidineand hypoxanthine; metabolic markers for which cells are selected, e.g.,their ability to grow on defined media containing the appropriate sugaras the sole carbon source, and markers which confer the ability of cellsto form colored colonies on chromogenic substrates or cause cells tofluoresce.

Among vectors suitable for use in bacteria include pQE70, pQE60, andpQE-9, available from QIAGEN, Inc., (Mississauga, Ontario, Canada); pBSvectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH6a, pNH18A,pNH46A, available from Stratagene (La Jolla, Calif.); and ptrc99a,pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia (Peapack,N.J.). Among suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL,available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Other suitable vectors include those employing a pTT vector backbone(Durocher et al. Nucl. Acids Res. 30 (2002)). Briefly, the pTT vectorbackbone may be prepared by obtaining pIRESpuro/EGFP (pEGFP) and pSEAPbasic vector(s), for example from Clontech (Palo Alto, Calif.), andpcDNA3.1, pcDNA3.1/Myc-(His)₆ and pCEP4 vectors can be obtained from,for example, Invitrogen. SuperGlo GFP variant (sgGFP) can be obtainedfrom Q-Biogene (Carlsbad, Calif.). Preparing a pCEP5 vector can beaccomplished by removing the CMV promoter and polyadenylation signal ofpCEP4 by sequential digestion and self-ligation using SalI and XbaIenzymes resulting in plasmid pCEP4Δ. A GblII fragment from pAdCMV5(Massie et al., J. Virol., 72: 2289-2296 (1998)), encoding theCMV5-poly(A) expression cassette may be ligated in BglII-linearizedpCEP4Δ, resulting in pCEP5 vector. The pTT vector can be prepared bydeleting the hygromycin (BsmI and SalI excision followed by fill-in andligation) and EBNA1 (ClaI and NsiI excision followed by fill-in andligation) expression cassettes. The ColEI origin (FspI-SalI fragment,including the 3′ end of β-lactamase ORF) can be replaced with aFspI-SalI fragment from pcDNA3.1 containing the pMBI origin (and thesame 3′ end of β-lactamase ORF). A Myc-(His)₆ C-terminal fusion tag canbe added to SEAP (HindIII-HpaI fragment from pSEAP-basic) followingin-frame ligation in pcDNA3.1/Myc-His digested with HindIII and EcoRV.Plasmids can subsequently be amplified in E. coli (DH5a) grown in LBmedium and purified using MAXI prep columns (Qiagen, Mississauga,Ontario, Canada). To quantify, plasmids can be subsequently diluted in50 mM Tris-HCl pH 7.4 and absorbencies can be measured at 260 nm and 280nm. Plasmid preparations with A₂₆₀/A₂₈₀ ratios between about 1.75 andabout 2.00 are suitable.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptides may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide.

The addition of peptide moieties to polypeptides to engender secretionor excretion, to improve stability and to facilitate purification, amongothers, are familiar and routine techniques in the art. A suitablefusion protein may comprise a heterologous region from immunoglobulinthat is useful to stabilize and purify proteins. For example, EP-A-O 464533 (Canadian counterpart 2045869) discloses fusion proteins containingvarious portions of constant region of immunoglobulin molecules togetherwith another human protein or part thereof. In many cases, the Fc partin a fusion protein is thoroughly advantageous for use in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected, and purified in the advantageous mannerdescribed. This is the case when the Fc portion proves to be a hindranceto use in therapy and diagnosis, for example when the fusion protein isto be used as an antigen for immunizations. In drug discovery, forexample, human proteins, such as hIL-5, have been fused with Fc portionsfor the purpose of high-throughput screening assays to identifyantagonists. See, Bennett et al., J. Molec. Recog., 8:52-58 (1995) andJohanson et al, J. Biol. Chem., 270:9459-9471 (1995).

The SDF-1 polypeptides can be recovered and purified from recombinantcell cultures by well-known methods, including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,and lectin chromatography. High performance liquid chromatography (HPLC)can be employed for purification. Polypeptides of the present inventioninclude products purified from natural sources, including bodily fluids,tissues and cells, whether directly isolated or cultured; products ofchemical synthetic procedures; and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect, and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in eukaryotic cells. While the N-terminal methionine on mostproteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Typically, a heterologous polypeptide, whether modified or unmodified,may be expressed as described above, or as a fusion protein, and mayinclude not only secretion signals, but also a secretory leadersequence. A secretory leader sequence of the invention directs certainproteins to the endoplasmic reticulum (ER). The ER separates themembrane-bound proteins from other proteins. Once localized to the ER,proteins can be further directed to the Golgi apparatus for distributionto vesicles; including secretory vesicles; the plasma membrane,lysosomes, and other organelles.

Proteins targeted to the ER by a secretory leader sequence can bereleased into the extracellular space as a secreted protein. Forexample, vesicles containing secreted proteins can fuse with the cellmembrane and release their contents into the extracellular space—aprocess called exocytosis. Exocytosis can occur constitutively or afterreceipt of a triggering signal. In the latter case, the proteins may bestored in secretory vesicles (or secretory granules) until exocytosis istriggered. Similarly, proteins residing on the cell membrane can also besecreted into the extracellular space by proteolytic cleavage of a“linker” holding the protein to the membrane.

Additionally, peptide moieties and/or purification tags may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability, and to facilitate purification, among other reasons, arefamiliar and routine techniques in the art. Suitable purification tagsinclude, for example, V5, HISX6, HISX8, avidin, and biotin.

The invention provides a fusion protein comprising a heterologous regionfrom an immunoglobulin that is useful to stabilize and purify proteins.For example, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins containing various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part of a fusion protein is advantageousfor use in therapy and diagnosis and thus results, for example, inimproved pharmacokinetic properties (EP-A 0232 262). On the other hand,for some uses it would be desirable to be able to delete the Fc partafter the fusion protein has been expressed, detected, and purified inthe advantageous manner described. This is the case when the Fc portionproves to be a hindrance to use in therapy and/or diagnosis, forexample, when the fusion protein is to be used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,Bennett et al., J. Molec. Recog., 8:52-58 (1995) and Johanson et al, J.Biol. Chem., 270:9459-9471 (1995).

A heterologous polypeptide of the invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, and highperformance liquid chromatography (HPLC). Polypeptides of the presentinvention include products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells, or from a cell free expression system. Depending upon the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

Polypeptides and Fragments

The invention further provides isolated SDF-1 polypeptides containingthe amino acid sequences encoded by the nucleotide sequences set forthin the Sequence Listing, the amino acid sequences set forth in theSequence Listing, and polypeptides comprising a fragment of any ofthese.

The invention provides secreted proteins, which are capable of beingdirected to the endoplasmic reticulum (ER), secretory vesicles, or theextracellular space as a result of a secretory leader, signal peptide,or leader sequence, as well as proteins released into the extracellularspace without necessarily containing a signal sequence. If a secretedprotein is released into the extracellular space, it may undergoextracellular processing to a mature polypeptide. Release into theextracellular space can occur by many mechanisms, including exocytosisand proteolytic cleavage.

The sequences of the invention encompass a variety of different types ofnucleic acids and polypeptides with different structures and functions.They can encode or comprise polypeptides belonging to different proteinfamilies (Pfam). The “Pfam” system is an organization of proteinsequence classification and analysis, based on conserved proteindomains; it can be publicly accessed in a number of ways, for example,at http://pfam.wustl.edu. Protein domains are portions of proteins thathave a tertiary structure and sometimes have enzymatic or bindingactivities; multiple domains can be connected by flexible polypeptideregions within a protein. Pfam domains can comprise the N-terminus orthe C-terminus of a protein, or can be situated at any point in between.The Pfam system identifies protein families based on these domains andprovides an annotated, searchable database that classifies proteins intofamilies (Bateman et al., Nucl. Acids Res. 30:276-280 (2002)). Sequencesof the invention can encode or be comprised of more than one Pfam.

Sequences of the invention may comprise an IL8 Pfam domain, as furtherdescribed below. Interleukin 8 (IL-8) is a chemokine that has beenreported, inter alia, to play a role in metastatic human cancer (Xie,Cytokine Growth Factor Rev. 12(4):375-391 (2001)) and in the airwayepithelium (Strieter, Am. J. Physiol. Lung Cell Mol. Physiol.283(4):L688-689 (2002). The IL8 Pfam includes a number of IL-8-likesecreted growth factors and interferons involved in mitogenic,chemotactic, and inflammatory activity. Members of the Pfam aregenerally characterized by a structure containing two conserveddisulfide bonds (http://pfam.wustl.edu/cgi-bin/getdesc?name=IL8).

Variant and Mutant Polypeptides

Protein engineering may be employed to improve or alter thecharacteristics of SDF-1 polypeptides of the invention. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or “muteins” including single or multiple amino acidsubstitutions, deletions, additions, or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984-2988(1993), reported modified KGF proteins that had heparin binding activityeven if 3, 8, or 27 amino-terminal amino acid residues were missing.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature from of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. Accordingly, the present invention further providespolypeptides having one or more residues deleted from the amino terminusof the amino acid sequences of the SDF-1 molecules as shown in theSequence Listing.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamma increases in activityas much as ten fold when 8-10 amino acid residues are deleted from thecarboxy terminus of the protein, see, for example, Dobeli et al., J.Biotechnology, 7:199-216 (1988).

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the SDF-1 polypeptides can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the SDF-1polypeptides which show substantial SDF-1 polypeptide activity or whichinclude regions of the SDF-1 proteins such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions, selected according to general rulesknown in the art, so as have little effect on activity. For example,guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al., Science, 247:1306-1310(1990), wherein the authors indicate that there are two main approachesfor studying the tolerance of an amino acid sequence to change. Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections, or screens, to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, et al., supra, and the referencescited therein. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gln, exchange of the basic residues Lys and Arg, andreplacements between the aromatic residues Phe and Tyr.

Thus, a fragment, derivative, or analog of a polypeptide of the SequenceListing or polypeptide encoded by a nucleic acid sequence of theSequence Listing may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue; such a substituted amino acid residue may or may not be oneencoded by the genetic code; (ii) one in which one or more of the aminoacid residues includes a substituent group; (iii) one in which themature polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol); or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide, a leader or secretory sequence, a sequence employed to purifythe above form of the polypeptide, or a proprotein sequence. Suchfragments, derivatives, and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Thus, the SDF-1 polypeptides of the present invention may include one ormore amino acid substitutions, deletions, or additions, either fromnatural mutations or human manipulation. As indicated, these changes maybe of a minor nature, such as conservative amino acid substitutions,that do not significantly affect the folding or activity of the protein.Conservative amino acid substitutions include the aromatic substitutionsPhe, Trp, and Tyr; the hydrophobic substitutions Leu, Iso, and Val; thepolar substitutions Glu and Asp; the basic substitutions Arg, Lys, andHis; the acidic substitutions Asp and Glu; and the small amino acidsubstations Ala, Ser, Thr, Met, and Gly.

Amino acids essential for the functions of SDF-1 polypeptides can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis, see, for example,Cunningham and Wells, Science, 244:1081-1085 (1989). The latterprocedure introduces single alanine mutations. The resulting mutantmolecules are then tested for biological activity such as receptorbinding, or in vitro or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because, for example, aggregatescan be immunogenic, Pinckard et al., Clin. Exp. Immunol., 2:331-340(1967); Robbins et al., Diabetes, 36:838-845 (1987); Cleland et al.,Crit. Rev. Therapeutic Drug Carrier Systems, 10:307-377 (1993).

Replacing amino acids can also change the selectivity of the binding ofa ligand to cell surface receptors. For example, Ostade et al., Nature,361:266-268 (1993) describes certain mutations resulting in selectivebinding of TNF-α to only one of the two known types of TNF receptors.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance, or photoaffinity labeling, for example, Smith etal., J. Mol. Biol., 224:899-904 (1992) and de Vos et al., Science,255:306-312 (1992).

The polypeptides of the present invention can be provided in an isolatedform, and can be substantially purified. A recombinantly producedversion of the herein described SDF-1 polypeptides can be substantiallypurified, e.g., by the one-step method described in Smith and Johnson,Gene, 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-SDF-1 antibodiesof the invention using methods which are well known in the art ofprotein purification. The polypeptides herein may be purified orisolated in the presence of ions or agents that aid in the refolding ofthe molecules or aid in dimerizing or trimerizing the molecules asconventional in the art.

Further polypeptides of the present invention include polypeptides whichhave at least 93%, 95%, 96%, 97%, 98%, or 99% similarity to thosedescribed above. The polypeptides of the invention also contain thosewhich are at least 93%, 94%, or 95%, 96%, 97%, 98%, or 99% identical toa polypeptide encoded by a nucleic acid sequence of the SequenceListing.

The % similarity of two polypeptides can be measured by a similarityscore determined by comparing the amino acid sequences of the twopolypeptides using the Bestfit program with the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) tofind the best segment of similarity between two sequences.

A polypeptide having an amino acid sequence at least, for example, 95%identical to a reference amino acid sequence of an SDF-1 polypeptide isone in which the amino acid sequence of the polypeptide is identical tothe reference sequence except that the polypeptide sequence may includeup to five amino acid alterations per each 100 amino acids of thereference polypeptide. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids, up to 5% of the total amino acid residues in the referencesequence, may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence, or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least93%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, an aminoacid sequence or to a polypeptide sequence encoded by a nucleic acidsequence set forth in the Sequence Listing can be determinedconventionally using known computer programs, such the Bestfit program.When using Bestfit or other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, that the percentage of identity is calculated overthe full length of the reference amino acid sequence and that gaps inhomology of up to 5% of the total number of amino acid residues in thereference sequence are allowed.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in assays for detecting SDF-1 protein expression, also asdescribed below, or as agonists and/or antagonists capable of enhancingor inhibiting SDF-1 protein function. These polypeptides can also beused in a yeast two-hybrid system to capture SDF-1 protein bindingproteins, which are also candidate agonists and antagonists, accordingto the present invention. The yeast two hybrid system is described inFields and Song, Nature, 340:245-246 (1989).

Aptamers

Another suitable agent for modulating an activity of a subjectpolypeptide is an aptamer. Aptamers of the invention include bothnucleotide and peptide aptamers. Nucleotide aptamers of the inventioninclude double stranded DNA and single stranded RNA molecules that bindto ADAM12 proteins or fragments thereof. Peptide aptamers are peptidesor small polypeptides that act as dominant inhibitors of proteinfunction. Peptide aptamers specifically bind to target proteins,blocking their functional ability (Kolonin et al., Proc. Natl. Acad.Sci. 95:14,266-14,271 (1998)).

Due to the highly selective nature of peptide aptamers, they can be usednot only to target a specific protein, but also to target specificfunctions of a given protein (for example, a signaling function).Further, peptide aptamers can be expressed in a controlled fashion byuse of promoters which regulate expression in a temporal, spatial, orinducible manner. Peptide aptamers act dominantly, therefore, they canbe used to analyze proteins for which loss-of-function mutants are notavailable. Aptamers of the invention may bind nucleotide cofactors(Latham et al., Nucl. Acids Res. 22:2817-2822 (1994)).

Peptide aptamers that bind with high affinity and specificity to atarget protein can be isolated by a variety of techniques known in theart. Peptide aptamers can be isolated from random peptide libraries byyeast two-hybrid screens (Xu et al., Proc. Natl. Acad. Sci.94:12,473-12,478. (1997)). They can also be isolated from phagelibraries (Hoogenboom et al., Immunotechnology 4:1-20 (1998)) orchemically generated peptides/libraries.

Epitope-Bearing Portions

In another aspect, the invention provides a polypeptide comprising anepitope-bearing portion of a polypeptide of the invention. The epitopeof this polypeptide portion is an immunogenic or antigenic epitope of apolypeptide of the invention. Immunogenic epitopes are those parts of aprotein that elicit an antibody response when the whole protein isprovided as the immunogen. On the other hand, a region of a proteinmolecule to which an antibody can bind is an antigenic epitope. Thenumber of immunogenic epitopes of a protein generally is less than thenumber of antigenic epitopes. See, for instance, Geysen et al., Proc.Natl. Acad. Sci., USA 81:3998-4002 (1983).

As to the selection of polypeptides bearing an antigenic epitope (i.e.,that contain a region of a protein molecule to which an antibody canbind), it is well known in that art that relatively short syntheticpeptides that mimic part of a protein sequence are routinely capable ofeliciting an antiserum that reacts with the partially mimicked protein.See, for instance, Sutcliffe et al., Science, 219:660-666 (1983).Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Antigenic epitope-bearingpeptides and polypeptides of the invention are therefore useful forraising antibodies, including monoclonal antibodies, that bindspecifically to a polypeptide of the invention. See, for instance,Wilson et al., Cell, 37:767-778 (1984). The epitope-bearing peptides andpolypeptides of the invention may be produced by any conventional means.See, for example, Houghten, Proc. Natl. Acad. Sci., USA 82:5131-5135(1985), and U.S. Pat. No. 4,631,211 (1986).

Epitope-bearing peptides and polypeptides of the invention can be usedto induce antibodies according to methods well known in the art. See,for instance, Bittle, et al, J. Gen. Virol., 66:2347-2354 (1985).Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art.See, for instance, U.S. Pat. No. 5,194,392 (1990), which describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 (1989) describes a method ofdetecting or determining a sequence of monomers which is a topographicalequivalent of a ligand which is complementary to the ligand binding siteof a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971(1996) discloses linear C1-C7-alkyl peralkylated oligopeptides, and setsand libraries of such peptides, as well as methods for using sucholigopeptide sets and libraries for determining the sequence of aperalkylated oligopeptide that preferentially binds to an acceptormolecule of interest. Thus, non-peptide analogs of the epitope-bearingpeptides of the invention also can be made routinely by these methods.

Fusion Proteins

As one of skill in the art will appreciate, SDF-1 polypeptides of thepresent invention, and the epitope-bearing fragments thereof describedabove, can be combined with parts of the constant domain ofimmunoglobulins, resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins, for example, EP A 394,827; Traunecker et al., Nature,331:84-86 (1988). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric SDF-1 protein or proteinfragment alone, for example, as described by Fountoulakis et al., J.Biochem., 270:3958-3964 (1995). Suitable chemical moieties forderivatization of a heterologous polypeptide include, for example,polymers, such as water soluble polymers, all or part of human serumalbumin, fetuin A, fetuin B, leucine zipper nuclear factor erythroidderivative-2 (NFE2), neuroretinal leucine zipper, mannose motif (mbp1),tetranectin, and an Fc region.

Polymers, e.g., water soluble polymers, are useful in the presentinvention as the polypeptide to which each polymer is attached will notprecipitate in an aqueous environment, such as a physiologicalenvironment. Polymers employed in the invention will be pharmaceuticallyacceptable for the preparation of a therapeutic product or composition.One skilled in the art will be able to select the desired polymer basedon such considerations as whether the polymer/protein conjugate will beused therapeutically and, if so, the desired dosage, circulation time,and resistance to proteolysis.

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers(PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll, ordextran and mixtures thereof.

As used herein, polyethylene glycol (PEG) is meant to encompass any ofthe forms that have been used to derivatize other proteins, such asmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water.

Specifically, a modified heterologous polypeptide of the invention maybe prepared by attaching polyaminoacids or branch point amino acids tothe polypeptide. For example, the polyaminoacid may be a carrier proteinthat serves to increase the circulation half life of the polypeptide(i.e., in addition to the advantages achieved via a fusion molecule).For the therapeutic purpose of the present invention, suchpolyaminoacids should ideally be those that have or do not createneutralizing antigenic response, or other adverse responses. Suchpolyaminoacids may be selected from serum album (such as human serumalbumin), an additional antibody or portion thereof, for example the Fcregion, fetuin A, fetuin B, leucine zipper nuclear factor erythroidderivative-2 (NFE2), neuroretinal leucine zipper, mannose motif (mbp1),tetranectin, or other polyaminoacids, e.g. lysines. As described herein,the location of attachment of the polyaminoacid may be at theN-terminus, or C-terminus, or other places in between, and also may beconnected by a chemical “linker” moiety to the selected molecule.

Polymers used herein, for example water soluble polymers, may be of anymolecular weight and may be branched or unbranched. The polymers eachtypically have an average molecular weight of between about 2 kDa toabout 100 kDa (the term “about” indicating that in preparations of apolymer, some molecules will weigh more, some less, than the statedmolecular weight). The average molecular weight of each polymer may bebetween about 5 kDa and about 50 kDa, or between about 12 kDa and about25 kDa. Generally, the higher the molecular weight or the more branches,the higher the polymer:protein ratio. Other sizes may also be used,depending on the desired therapeutic profile; for example, the durationof sustained release; the effects, if any, on biological activity; theease in handling; the degree or lack of antigenicity; and other knowneffects of a polymer on a modified molecule of the invention.

Polymers employed in the present invention are typically attached to aheterologous polypeptide with consideration of effects on functional orantigenic domains of the polypeptide. In general, chemicalderivatization may be performed under any suitable condition used toreact a protein with an activated polymer molecule. Activating groupswhich can be used to link the polymer to the active moieties include thefollowing: sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate,azidirine, oxirane, and 5-pyridyl.

Polymers of the invention are typically attached to a heterologouspolypeptide at the alpha (α) or epsilon (ε) amino groups of amino acidsor a reactive thiol group, but it is also contemplated that a polymergroup could be attached to any reactive group of the protein that issufficiently reactive to become attached to a polymer group undersuitable reaction conditions. Thus, a polymer may be covalently bound toa heterologous polypeptide via a reactive group, such as a free amino orcarboxyl group. The amino acid residues having a free amino group mayinclude lysine residues and the N-terminal amino acid residue. Thosehaving a free carboxyl group may include aspartic acid residues,glutamic acid residues, and the C-terminal amino acid residue. Thosehaving a reactive thiol group include cysteine residues.

Methods for preparing fusion molecules conjugated with polymers, such aswater soluble polymers, will each generally involve (a) reacting aheterologous polypeptide with a polymer under conditions whereby thepolypeptide becomes attached to one or more polymers and (b) obtainingthe reaction product. Reaction conditions for each conjugation may beselected from any of those known in the art or those subsequentlydeveloped, but should be selected to avoid or limit exposure to reactionconditions such as temperatures, solvents, and pH levels that wouldinactivate the protein to be modified. In general, the optimal reactionconditions for the reactions will be determined case-by-case based onknown parameters and the desired result. For example, the larger theratio of polymer:polypeptide conjugate, the greater the percentage ofconjugated product. The optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted polypeptide or polymer)may be determined by factors such as the desired degree ofderivatization (e.g., mono-, di-tri- etc.), the molecular weight of thepolymer selected, whether the polymer is branched or unbranched and thereaction conditions used. The ratio of polymer (e.g., PEG) to apolypeptide will generally range from 1:1 to 100:1. One or more purifiedconjugates may be prepared from each mixture by standard purificationtechniques, including among others, dialysis, salting-out,ultrafiltration, ion-exchange chromatography, gel filtrationchromatography, and electrophoresis.

One may specifically desire an N-terminal chemically modified protein.One may select a polymer by molecular weight, branching, etc., theproportion of polymers to protein (polypeptide or peptide) molecules inthe reaction mix, the type of reaction to be performed, and the methodof obtaining the selected N-terminal chemically modified protein. Themethod of obtaining the N-terminal chemically modified proteinpreparation (i.e., separating this moiety from other monoderivatizedmoieties if necessary) may be by purification of the N-terminalchemically modified protein material from a population of chemicallymodified protein molecules.

Selective N-terminal chemical modification may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, substantially selective derivatization of theprotein at the N-terminus with a carbonyl group containing polymer isachieved. For example, one may selectively attach a polymer to theN-terminus of the protein by performing the reaction at a pH whichallows one to take advantage of the pKa differences between the e-aminogroup of the lysine residues and that of the α-amino group of theN-terminal residue of the protein. By such selective derivatization,attachment of a polymer to a protein is controlled: the conjugation withthe polymer takes place predominantly at the N-terminus of the proteinand no significant modification of other reactive groups, such as thelysine side chain amino groups, occurs. Using reductive alkylation, thepolymer may be of the type described above and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may also beused.

In one embodiment, the present invention contemplates the chemicallyderivatized polypeptide to include mono- or poly- (e.g., 2-4) PEGmoieties. Pegylation may be carried out by any of the pegylationreactions known in the art. Methods for preparing a pegylated proteinproduct will generally include (a) reacting a polypeptide withpolyethylene glycol (such as a reactive ester or aldehyde derivative ofPEG) under conditions whereby the protein becomes attached to one ormore PEG groups; and (b) obtaining the reaction product(s). In general,the optimal reaction conditions for the reactions will be determinedcase by case based on known parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art. See, for example, EP 0 401 384; Malik et al., Exp. Hematol.,20:1028-1035 (1992); Francis, Focus on Growth Factors, 3(2):4-10 (1992);EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and the otherpublications cited herein that relate to pegylation, the disclosures ofwhich are hereby incorporated by reference.

Additionally, heterologous polypeptides of the present invention and theepitope-bearing fragments thereof described herein can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These particular fusion molecules facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins, for example,EP A 394,827; Traunecker et al., Nature, 331:84-86 (1988). Fusionmolecules that have a disulfide-linked dimeric structure due to the IgGpart can also be more efficient in binding and neutralizing othermolecules than, for example, a monomeric polypeptide or polypeptidefragment alone, see, for example, Fountoulakis et al., J. Biochem.,270:3958-3964 (1995).

In another described embodiment, a human serum albumin fusion moleculemay also be prepared as described herein and as further described inU.S. Pat. No. 6,686,179.

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide that facilitates purification of thefused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide such as the tag provided in a pQEvector (QIAGEN, Inc., among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Another peptide tag useful forpurification, the chemagglutinin HA tag, corresponds to an epitopederived from the influenza hemagglutinin protein. (Wilson et al., Cell37:767 (1984)).

Secretory Leader Sequences

As demonstrated herein, and in U.S. 60/647,013, in order for somesecreted proteins to express and secrete in larger quantities, asecretory leader sequence from another, i.e., different, secretedprotein is desirable. Employing heterologous secretory leader sequencesis advantageous in that a resulting mature amino acid sequence, i.e.,protein, of the secreted polypeptide is not altered as the secretoryleader sequence is removed in the ER during the secretion process.Moreover, the addition of a heterologous secretory leader is oftenrequired to express and secrete, for example, extracellular domains ofType II single transmembrane proteins (STM), as the secretory leader,which is also a transmembrane spanning domain, must typically be removedso that they may be soluble.

Co-Translational and Post-Translational Modifications

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand. Any of numerous chemical modifications may be carriedout by known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease; NABH₄; acetylation; formylation; oxidation; reduction; and/ormetabolic synthesis in the presence of tunicamycin.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic, or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability, and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three, or more attachedchemical moieties.

A polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Suitable fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

Compositions

In some embodiments, SDF-1 compositions are provided in formulation withpharmaceutically acceptable excipients, a wide variety of which areknown in the art (Gennaro, Remington: The Science and Practice ofPharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003);Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems,7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe et al.,Handbook of Pharmaceutical Excipients, 3^(rd) ed., Pharmaceutical Press(2000)). Pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

In pharmaceutical dosage forms, the compositions of the invention can beadministered in the form of their pharmaceutically acceptable salts, orthey can also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The subjectcompositions are formulated in accordance to the mode of potentialadministration. Administration of the agents can be achieved in variousways, including oral, buccal, nasal, rectal, parenteral,intraperitoneal, intradermal, transdermal, subcutaneous, intravenous,intra-arterial, intracardiac, intraventricular, intracranial,intratracheal, and intrathecal administration, etc., or otherwise byimplantation or inhalation. Thus, the subject compositions can beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, enemas, injections, inhalants and aerosols.The following methods and excipients are merely exemplary and are in noway limiting.

Compositions for oral administration can form solutions, suspensions,tablets, pills, granules, capsules, sustained release formulations, oralrinses, or powders. For oral preparations, the agents, polynucleotides,and polypeptides can be used alone or in combination with appropriateadditives, for example, with conventional additives, such as lactose,mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art (Gennaro, 2003). The compositionor formulation to be administered will, in any event, contain a quantityof the agent adequate to achieve the desired state in the subject beingtreated.

The agents, polynucleotides, and polypeptides can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. Other formulations for oral orparenteral delivery can also be used, as conventional in the art.

The antibodies, agents, polynucleotides, and polypeptides can beutilized in aerosol formulation to be administered via inhalation. Thecompounds of the present invention can be formulated into pressurizedacceptable propellants such as dichlorodifluoromethane, propane,nitrogen, and the like. Further, the agent, polynucleotides, orpolypeptide composition may be converted to powder form foradministration intranasally or by inhalation, as conventional in theart.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

A polynucleotide, polypeptide, or other modulator, can also beintroduced into tissues or host cells by other routes, such as viralinfection, microinjection, or vesicle fusion. For example, expressionvectors can be used to introduce nucleic acid compositions into a cellas described above. Further, jet injection can be used for intramuscularadministration (Furth et al., Anal. Biochem. 205:365-368 (1992)). TheDNA can be coated onto gold microparticles, and delivered intradermallyby a particle bombardment device, or “gene gun” as described in theliterature (Tang et al., Nature 356:152-154 (1992)), where goldmicroprojectiles are coated with the DNA, then bombarded into skincells.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions can be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet, or suppository, contains apredetermined amount of the composition containing one or more agents.Similarly, unit dosage forms for injection or intravenous administrationcan comprise the agent(s) in a composition as a solution in sterilewater, normal saline or another pharmaceutically acceptable carrier.

Chromosome Assays

In certain embodiments relating to chromosomal mapping, a cDNA hereindisclosed is used to clone the genomic nucleic acid of the SDF-1. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are commercially available. The genomic DNAthen is used for in situ chromosome mapping using techniques well knownfor this purpose. Therefore, the nucleic acid molecules of the presentinvention are also valuable for chromosome identification. The sequenceis specifically targeted to and can hybridize with a particular locationon an individual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primersfrom the cDNA. Computer analysis of the 3′ untranslated region is usedto rapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primersare then used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphaseChromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with a cDNA as short asapproximately 50-60 bases. For a review of this technique, see Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

Next, differences can be determined in the cDNA or genomic sequences ofaffected and unaffected individuals. If a mutation is observed in someor all of the affected individuals but not in any normal individuals,then the mutation is likely to be the causative agent of the disease.With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes (assuming 1 megabase mapping resolution and one gene per20 kb).

Using methods described above, the SDF-1 gene of the invention has beenmapped by fluorescent in situ hybridization to human chromosome 10q11.1.The corresponding map position in the mouse includes several diseaseloci, including whim syndrome, immunologic deficiency syndromes, HIVinfections, inflammation, acquired immunodeficiency syndrome, B cellleukemias, chronic lymphocytic leukemia, metastasis, and rheumatoidarthritis (http://biostatpub2.mdanderson.org/cgi-bin/genecards/carddisp?CXCL12&search=sdf-1&suff=txt).

Identification of Agonists and Antagonists

This invention provides modulators, i.e., polypeptides, polynucleotides,or other agents that increase or decrease the activity of their target.They may act as an agonist or antagonist, and interfere with the bindingor activity of polypeptides or polynucleotides. Such modulators oragents include, for example, polypeptide variants, whether agonist orantagonist; antibodies, whether agonist or antagonist; solublereceptors, usually antagonists; small molecule drugs, whether agonist orantagonist; RNAi, usually an antagonist; antisense molecules, usually anantagonist; and ribozymes, usually an antagonist. In some embodiments,an agent is a subject polypeptide, where the subject polypeptide itselfis administered to an individual. In some embodiments, an agent is anantibody specific for a subject “target” polypeptide. In someembodiments, an agent is a chemical compound such as a small moleculethat may be useful as an orally available drug. Such modulation includesthe recruitment of other molecules that directly effect the modulation.For example, an antibody that modulates the activity of a subjectpolypeptide that is a receptor on a cell surface may bind to thereceptor and fix complement, activating the complement cascade andresulting in lysis of the cell. An agent which modulates a biologicalactivity of a subject polypeptide or polynucleotide increases ordecreases the activity or binding at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 50%, atleast about 80%, or at least about 2-fold, at least about 5-fold, or atleast about 10-fold or more when compared to a suitable control.

This invention also provides a method of screening compounds to identifythose which modulate the action of the polypeptide of the presentinvention. An example of such an assay comprises combining a mammalianfibroblast cell and the polypeptide(s) of the present invention, thecompound to be screened and ³[H] thymidine under cell culture conditionswhere the fibroblast cell would normally proliferate. A control assaymay be performed in the absence of the compound to be screened andcompared to the amount of fibroblast proliferation in the presence ofthe compound to determine if the compound stimulates proliferation bydetermining the uptake of ³[H] thymidine in each case. The amount offibroblast cell proliferation is measured by liquid scintillationchromatography, which measures the incorporation of ³[H] thymidine. Bothagonistic and antagonistic compounds may be identified by thisprocedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention, as described above,is incubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the SDF-1 receptor is measured and theability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems include,but are not limited to, cAMP, guanylate cyclase, ion channels, andphosphoinositide hydrolysis.

Examples of antagonistic compounds include antibodies, or in some cases,oligonucleotides, which bind to a receptor of a polypeptide of thepresent invention but elicit no second messenger response, or which bindto the SDF-1 polypeptide itself. Alternatively, a potential antagonistmay be a mutant form of the polypeptide which binds to the receptors butelicits no second messenger response, thus effectively blocking theaction of the polypeptide.

Another compound antagonistic to SDF-1 genes and gene products is anantisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA; both methods are based on the bindingof a polynucleotide to DNA or RNA. For example, a 5′ coding portion ofthe polynucleotide sequence, which encodes mature polypeptides of thepresent invention, can be used to design an antisense RNAoligonucleotide of from about 10 to about 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription, for example, a triple helix—see Lee et al.,Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);and Dervan et al., Science, 251: 1360 (1991), thereby preventingtranscription and the production of the polypeptides of the presentinvention. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the polypeptide,as described by Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). The oligonucleotides described above canalso be delivered to cells such that the antisense RNA or DNA isexpressed in vivo to inhibit polypeptide production.

Potential antagonist compounds also include small molecules which bindto and occupy the binding site of the receptors, thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules. Antagonistcompounds may be employed to inhibit the cell growth and proliferationeffects of the polypeptides of the present invention on neoplastic cellsand tissues, i.e. stimulation of angiogenesis of tumors, and, therefore,retard or prevent abnormal cellular growth and proliferation, forexample, in tumor formation or growth.

The present invention also provides methods for identifying agents, suchas antibodies, which enhance or block the actions of SDF-1 molecules oncells. For example these agents may enhance or block interaction ofSDF-1-binding molecules, such as receptors. Agents of interest includeboth agonists and antagonists. The invention provides agonists whichincrease the natural biological functions of SDF-1 or which function ina manner similar to SDF-1. The invention also provides antagonists,which decrease or eliminate the functions of SDF-1.

One method of identifying SDF-1 agonists and antagonists involvesbiochemical assays following subcellular fractionation. For example, acellular compartment, such as a membrane or cytosolic preparation may beprepared from a cell that expresses a molecule that binds SDF-1molecules, such as a molecule of a signaling or regulatory pathwaymodulated by SDF-1 molecules. Subcellular fractionation methods areknown in the art of cell biology, and can be tailored to produce crudefractions with discrete and defined components, e.g., organelles ororganellar membranes. The preparation is incubated with labeled SDF-1molecules in the absence or the presence of a candidate molecule whichmay be an SDF-1 agonist or antagonist. The ability of the candidatemolecule to interact with the binding molecule or an SDF-1 molecules isreflected in decreased binding of the labeled ligand. Molecules whichbind gratuitously, that is, without inducing the effects of SDF-1molecules, are most likely antagonists. Molecules that bind well andelicit effects that are the same as or closely related to SDF-1molecules may potentially prove to be agonists.

The effects of potential agonists and antagonists may by measured, forinstance, by determining an activity of one or more components of asecond messenger system following interaction of the candidate moleculewith a cell or appropriate cell preparation, and comparing the effectwith that of SDF-1 molecules, or with that of molecules that elicit thesame effects as SDF-1. Second messenger systems which may be useful inthis regard include, but are not limited to, cAMP, cGMP, ion channel,and phosphoinositide hydrolysis second messenger systems.

Another example of an assay for the identification of SDF-1 antagonistsis a competitive assay that combines a mixture of SDF-1 molecules and apotential antagonist, with membrane-bound SDF-1 receptor molecules.Under appropriate conditions for a competitive inhibition assay, thisassay can also be performed with recombinant SDF-1 receptor molecules.SDF-1 molecules can be labeled, such as by radioactivity, such that thenumber of SDF-1 molecules bound to a receptor molecule can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, polypeptides, andantibodies that bind to a polypeptide of the invention, and therebyinhibit or extinguish its activity. Potential antagonists also may besmall organic molecules, polypeptides such as closely related proteinsor antibodies that bind the same sites on a binding molecule, such as areceptor molecule, without inducing SDF-1-induced activities, therebypreventing the action of SDF-1 molecules by excluding SDF-1 moleculesfrom binding. Antagonists of the invention include fragments of theSDF-1 molecules having the nucleic acid and amino acid sequences shownin the Sequence Listing.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through, e.g.,antisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1360 (1991). The methods are based on the binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to about 40 base pairs in length. A DNA oligonucleotideis designed to be complementary to a region of the gene involved intranscription, thereby preventing transcription and the subsequentproduction of SDF-1 molecules. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into an SDF-1 polypeptide. The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of SDF-1 molecules.

Therapeutic Uses of SDF-1 and its Agonists and Antagonists

SDF-1 polynucleotides, polypeptides, agonists, and/or antagonists of theinvention may be used in developing treatments for any disorder mediated(directly or indirectly) by defective SDF-1 molecules, or insufficientamounts of either of these. SDF-1 polypeptides, agonists, and/orantagonists may be administered to a patient (e.g., a mammal, such ashuman) afflicted with such a disorder. Alternatively, a gene therapyapproach may be applied to treat such disorders. Disclosure herein ofSDF-1 nucleotide sequences permits the detection of defective SDF-1genes, and the replacement thereof with normal SDF-1-encoding genes.Defective genes may be detected in in vitro diagnostic assays, and bycomparison of the SDF-1 nucleotide sequences disclosed herein with thatof an SDF-1 gene derived from a patient suspected of harboring a defectin this gene.

The SDF-1 molecules of the present invention may be employed to treatlymphoproliferative disease which results in lymphadenopathy. They mayalso mediate apoptosis by stimulating clonal deletion of T-cells andmay, therefore, be employed to treat autoimmune disease to stimulateperipheral tolerance and cytotoxic T-cell mediated apoptosis. The SDF-1molecules may further be employed as a research tool in elucidating thebiology of autoimmune disorders, including systemic lupus erythematosus(SLE), Graves' disease, immunoproliferative disease lymphadenopathy(IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblasticlymphadenopathy (IBL), rheumatoid arthritis, diabetes, and multiplesclerosis. It also finds use in treating allergies and graft versus hostdisease.

The SDF-1 polynucleotides, polypeptides, and/or agonists or antagonistsof the invention may also be used to treat, prevent, diagnose, and/orprognose diseases which include, but are not limited to, autoimmunedisorders, immunodeficiency disorders, and graft versus host disease.Specific types of autoimmune diseases that can be treated with themolecules of the invention include, but are not limited to, Th2lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th-1 lymphocyte-relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjögren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes).

The SDF-1 polypeptides of the present invention may be employed toinhibit neoplasia, such as tumor cell growth. They may be responsiblefor tumor destruction through apoptosis and cytotoxicity to certaincells.

Diseases associated with increased cell survival, or the inhibition ofapoptosis, that may be treated, prevented, diagnosed and/or prognosedwith the SDF-1 polynucleotides, polypeptides and/or agonists orantagonists of the invention include, but are not limited to, cancers(such as follicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjögren's syndrome,Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus, and immune-related glomerulonephritis, autoimmunegastritis, autoimmune thrombocytopenic purpura, and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft vs. host disease (acute and/orchronic), acute graft rejection, and chronic graft rejection. SDF-1polynucleotides and/or polypeptides, and their agonists, and/orantagonists may be used to inhibit growth, progression, and/ormetastasis of cancers, in particular those listed above or in theparagraph that follows.

Additional diseases or conditions associated with increased cellsurvival, that may be treated, prevented, diagnosed, and/or prognosedwith the SDF-1 polynucleotides and/or polypeptides and their agonistsand/or antagonists include, but are not limited to, progression and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias, (e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenström'smacroglobulinemia, heavy chain diseases, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis, that may be treated,prevented, diagnosed and/or prognosed with the SDF-1 polynucleotides,polypeptides and/or agonists or antagonists of the invention include,but are not limited to, AIDS, neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa, cerebellar degeneration, and brain tumor or priorassociated disease); diabetes, autoimmune disorders (such as, multiplesclerosis, Sjögren's syndrome, Graves' disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, SLE, immune-related glomerulonephritis,autoimmune gastritis, thrombocytopenic purpura, and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graftvs. host disease (acute and/or chronic), ischemic injury (such as thatcaused by myocardial infarction, stroke, and reperfusion injury), liverinjury or disease (e.g., hepatitis related liver injury, cirrhosis,ischemia/reperfusion injury, cholestosis (bile duct injury) and livercancer); toxin-induced liver disease (such as that caused by alcohol),septic shock, ulcerative colitis, cachexia, and anorexia. In someembodiments, SDF-1 polynucleotides, polypeptides, agonists, and/orantagonists are used to treat the diseases and disorders listed above.

Molecules of the invention are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The molecules of the invention can beused accordingly in a variety of settings for the treatment of aninfectious disease in an animal. In the context of an infectiousdisease, the term “treating” includes any or all of preventing thegrowth, multiplication or replication of the pathogen that causes theinfectious disease and ameliorating one or more symptoms of aninfectious disease.

Many of the pathologies associated with HIV are mediated by apoptosis,including HIV-induced nephropathy and HIV encephalitis. Thus, in someembodiments, SDF-1 polynucleotides, polypeptides, agonists orantagonists of the invention are used to treat AIDS and pathologiesassociated with AIDS.

Another embodiment of the present invention is directed to the use ofSDF-1 polynucleotides, polypeptides, or antagonists to reduceSDF-1-mediated death of T cells in HIV-infected patients. The role of Tcell apoptosis in the development of AIDS has been the subject of anumber of studies (see, for example, Meyaard et al., Science,257:217-219 (1992); Groux et al., J. Exp. Med., 175:331 (1992); andOyaizu et al., in Cell Activation and Apoptosis in HIV Infection,Andrieu and Lu, Eds., Plenum Press, New York, pp. 101-114 (1995)).Fas-mediated apoptosis has been implicated in the loss of T cells in HIVpositive individuals (Katsikis et al., J. Exp. Med. 181:2029-2036(1995). It is also likely that T cell apoptosis occurs through multiplemechanisms.

Activated human T cells are induced to undergo programmed cell death(apoptosis) upon triggering through the CD3/T cell receptor complex, aprocess termed activated-induced cell death (AICD). AICD of CD4 T cellsisolated from HIV-infected asymptomatic individuals has been reported(Groux et al., supra). Thus, AICD may play a role in the depletion ofCD4+ T cells and the progression to AIDS in HIV-infected individuals.Thus, the present invention provides a method of inhibitingSDF-1-mediated T cell death in HIV patients, comprising administeringSDF-1 polynucleotides, polypeptides, or antagonists of the invention tothe patients. In one embodiment, the patient is asymptomatic whentreatment with SDF-1 polynucleotides, polypeptides, or antagonistscommences. If desired, prior to treatment, peripheral blood T cells maybe extracted from an HIV patient, and tested for susceptibility toSDF-1-mediated cell death by procedures known in the art. In oneembodiment, a patient's blood or plasma is contacted with SDF-1antagonists (e g., anti-SDF-1 antibodies) of the invention ex vivo. TheSDF-1 antagonists may be bound to a suitable chromatography matrix byprocedures known in the art. The patient's blood or plasma flows througha chromatography column containing SDF-1 antagonist bound to the matrix,before being returned to the patient. The immobilized SDF-1 antagonistbinds SDF-1, thus removing SDF-1 protein from the patient's blood.

In additional embodiments, an SDF-1 polynucleotide, polypeptide, orantagonist of the invention is administered in combination with otherinhibitors of T cell apoptosis. For example, as discussed above,Fas-mediated apoptosis also has been implicated in loss of T cells inHIV individuals (Katsikis et al., J. Exp. Med., 181:2029-2036 (1995)).Thus, a patient susceptible to both Fas ligand mediated andSDF-1-mediated T cell death may be treated with both an agent thatblocks SDF-1/SDF-1 receptor interactions and an agent that blocksFas-ligand/Fas interactions. Suitable agents for blocking binding ofFas-ligand to Fas include, but are not limited to, soluble Faspolypeptides; multimeric forms of soluble Fas polypeptides (e.g., dimersof sFas/Fc); anti-Fas antibodies that bind Fas without transducing thebiological signal that results in apoptosis; anti-Fas-ligand antibodiesthat block binding of Fas-ligand to Fas; and muteins of Fas-ligand thatbind Fas but do not transduce the biological signal that results inapoptosis. Preferably, the antibodies employed according to this methodare monoclonal antibodies. Examples of suitable agents for blockingFas-ligand/Fas interactions, including blocking anti-Fas monoclonalantibodies, are described in WO 95/10540, hereby incorporated byreference.

In another example, agents which block binding of SDF-1 to an SDF-1receptor are administered with the SDF-1 polynucleotides, polypeptides,or antagonists of the invention. Such agents include, but are notlimited to, soluble SDF-1 receptor polypeptides; multimeric forms ofsoluble SDF-1 receptor polypeptides; and SDF-1 receptor antibodies thatbind the SDF-1 receptor without transducing the biological signal thatresults in apoptosis, anti-SDF-1 antibodies that block binding of SDF-1to one or more SDF-1 receptors, and muteins of SDF-1 that bind SDF-1receptors but do not transduce the biological signal that results inapoptosis.

SDF-1 polypeptides of the invention may also be employed to regulatehematopoiesis and, in particular, erythropoiesis. Hematopoiesis is amulti-step cell proliferation and differentiation process which beginswith a pool of multipotent stem cells. These cells can proliferate anddifferentiate into hematopoietic progenitors in reply to differentstimuli. The SDF-1 polypeptides of the invention, as well as agonistsand antagonists thereof, may be used to either stimulate or inhibitdevelopment of hematopoietic cells and, in particular, erythropoieticprecursor cells.

SDF-1 may be used to treat B-cell deficiencies, including those arisingin the context of infection, cancer treatment, transplant,immunodeficiency, immune disorder, agammaglobulinemia,hypogammaglobulinemia, defects in B cell development, defects in B cellfunction, defects in B-cell regulation, and shortened B cell lifespan.IL-7 is one of the factors involved in mediating B cell development(Stoddart et al., Immunol. Rev. 175:47-58 (2000)). Specifically, SDF-1may be used to treat Bruton agammaglobulinemia, an X chromosome-linkedagammaglobulinemia conventionally understood as a life-threateningdisease that involves a failure in normal development of B lymphocytesand is associated with missense mutations in BTK, a gene encoding acytoplasmic tyrosine kinase (Bruton agammaglobulinemia tyrosine kinase,EC 2.7.1.112), a member of the Tec family of protein-tyrosine kinases(Ohta et al., Proc. Natl. Acad. Sci. 91:9062-9066 (1994).

SDF-1 may be used to treat platelet deficiencies, such as manifest bythrombocytopenia. It can stimulate the growth and proliferation oflymphocytes, and can promote angiogenesis. SDF-1 is useful in thetreatment of diabetes. It can modulate the immune response of anorganism, can treat allergies, and can be used to treat and/or preventinfection. SDF-1 can inhibit tumor growth and can be used to treatcancer.

SDF-1 can treat or prevent ischemic disease. In an embodiment, theinvention provides

Additionally, molecules of the invention may be employed as agents toboost immunoresponsiveness among individuals having a temporary immunedeficiency. Conditions resulting in a temporary immune deficiency thatmay be ameliorated or treated by administering the SDF-1 polypeptides orpolynucleotides of the invention, or agonists thereof, include, but arenot limited to, recovery from viral infections (e.g., influenza),conditions associated with malnutrition, recovery from infectiousmononucleosis, or conditions associated with stress, recovery frommeasles, recovery from blood transfusion, and recovery from surgery.

In the context of an autoimmune disease, the term “treating” includesany or all of preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells capable ofproducing an autoimmune antibody, lessening the autoimmune-antibodyburden, and ameliorating one or more symptoms of an autoimmune disease.

SDF-1 polynucleotides or polypeptides of the invention, or agonists orantagonists thereof, may be used to diagnose, prognose, treat, orprevent one or more of the following diseases or disorders, orconditions associated therewith: primary immunodeficiencies,immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrowtransplant (e.g., recent bone marrow transplant in adults or children),chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult orpediatric HIV infection), chronic inflammatory demyelinatingpolyneuropathy, and post-transfusion purpura.

Additionally, SDF-1 polynucleotides or polypeptides of the invention, oragonists or antagonists thereof, may be used to diagnose, prognose,treat or prevent one or more of the following diseases, disorders, orconditions associated therewith, Guillain-Barre syndrome, anemia (e.g.,anemia associated with parvovirus B19, patients with stable multiplemyeloma who are at high risk for infection (e.g., recurrent infection),autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolyticanemia), thrombocytopenia (e.g., neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (e.g., cytomegalovirus(CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia(e.g., hypogammaglobulinemic neonates with risk factor for infection ormorbidity), epilepsy (e.g., intractable epilepsy), systemic vasculiticsyndromes, myasthenia gravis (e.g., decompensation in myastheniagravis), dermatomyositis, and polymyositis.

Autoimmune disorders and conditions associated with these disorders thatmay be treated, prevented, and/or diagnosed with the SDF-1polynucleotides, polypeptides, and/or antagonist of the invention (e.g.,anti-SDF-1 antibodies), include, but are not limited to, autoimmunehemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathicthrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia,antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis,neuritis, uveitis ophthalmia, polyendocrinopathies, purpura (e.g.,Henloch-Scoenlein purpura), Reiter's disease, stiff-man syndrome,autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disease.

Additional autoimmune disorders highly likely to be treated, prevented,and/or diagnosed with the compositions of the invention include, but arenot limited to, autoimmune thyroiditis, hypothyroidism (i.e.,Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediatedand humoral thyroid cytotoxicity), systemic lupus erythematosus (oftencharacterized, e.g., by circulating and locally generated immunecomplexes), Goodpasture's syndrome (often characterized, e.g., byanti-basement membrane antibodies), pemphigus (often characterized,e.g., by epidermal acantholytic antibodies), receptor autoimmunitiessuch as, for example, (a) Graves' disease (often characterized, e.g., byTSH receptor antibodies), (b) myasthenia gravis (often characterized,e.g., by acetylcholine receptor antibodies), and (c) insulin resistance(often characterized, e.g., by insulin receptor antibodies), autoimmunehemolytic anemia (often characterized, e.g., by phagocytosis ofantibody-sensitized red blood cells), autoimmune thrombocytopenicpurpura (often characterized, e.g., by phagocytosis ofantibody-sensitized platelets.

Additional autoimmune disorders which may be treated, prevented, and/ordiagnosed with the compositions of the invention include, but are notlimited to, rheumatoid arthritis (often characterized, e.g., by immunecomplexes in joints), scleroderma with anti-collagen antibodies (oftencharacterized, e.g., by nucleolar and other nuclear antibodies), mixedconnective tissue disease (often characterized, e.g., by antibodies toextractable nuclear antigens (e.g., ribonucleoprotein)),polymyositis/dermatomyositis (often characterized, e.g., by nonhistoneanti-nuclear antibodies), pernicious anemia (often characterized, e.g.,by antibodies to parietal cells, microsomes, and intrinsic factor),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes) such as primary glomerulonephritis and IgAnephropathy, bullous pemphigoid (often characterized, e.g., by IgG andcomplement in the basement membrane), Sjögren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone anti-nuclear antibodies (SS-B)), diabetes mellitus (oftencharacterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

Further autoimmune disorders which may be treated, prevented, and/ordiagnosed with the compositions of the invention include, but are notlimited to, chronic active hepatitis (often characterized, e.g. bysmooth muscle antibodies), primary biliary cirrhosis (oftencharacterized, e.g., by mitochondrial antibodies), other endocrine glandfailure (often characterized, e.g., by specific tissue antibodies insome cases), vitiligo (often characterized, e.g., by melanocyteantibodies), vasculitis (often characterized, e.g., by Ig and complementin vessel walls and/or low serum complement), post-myocardial infarction(often characterized, e.g., by myocardial antibodies), cardiotomysyndrome (often characterized, e.g., by myocardial antibodies),urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE),atopic dermatitis (often characterized, e.g., by IgG and IgM antibodiesto IgE), asthma (often characterized, e.g., by IgG and IgM antibodies toIgE), inflammatory myopathies, and many other inflammatory,granulamatous, degenerative, and atrophic disorders.

In an additional embodiment, SDF-1 polynucleotides or polypeptides, orantagonists thereof (e.g., anti-SDF-1 antibodies) are used to treat orprevent systemic lupus erythematosus and/or diseases, disorders orconditions associated therewith. Lupus-associated diseases, disorders,or conditions that may be treated or prevented with SDF-1polynucleotides or polypeptides, or antagonists of the invention,include, but are not limited to, hematologic disorders (e.g., hemolyticanemia, leukopenia, lymphopenia, and thrombocytopenia), immunologicdisorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes,photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss,serositis (e.g., pleuritus (pleurisy)), renal disorders (e.g.,nephritis), neurological disorders (e.g., seizures, peripheralneuropathy, CNS related disorders), gastrointestinal disorders, Raynaudphenomenon, and pericarditis.

SDF-1 polypeptides, agonists, or antagonists of the invention may beused to treat diseases associated with ischemia, e.g., cardiovasculardisorders, including peripheral artery disease, such as limb ischemia.They may also include stroke, vascular disease, and fulminant liverfailure. In the context of an ischemic disease, the term “treating”includes any or all of preventing the growth, multiplication, orreplication of the pathogen that causes the ischemic disease andameliorating one or more symptoms of an ischemic disease.

Stem cell mobilization to the heart and differentiation into cardiacmyocytes is a naturally occurring process. Askari et al., Lancet362:697-703 (2005), have speculated that up-regulation of this processcan help recover myocardial function following infarction. In anembodiment, SDF-1 induces therapeutic stem cell homing to injuredmyocardium. SDF-1 has been observed to be up-regulated followingmyocardial infarction (Askari et al., supra). The invention providescompositions and methods for providing SDF-1 to the heart. For example,autologous cells genetically modified with molecules of the SequenceListing can be transplanted to a subject that can benefit from suchcells. Bone marrow stimulation may be performed in conjunction with thetransplantation. Cardiac fibroblasts and cardiac myoblasts can betransfected with SDF-1 as described by Askari et al., supra, or othermeans known in the art, and provided in vivo by local administration. Arodent model with a ligation of the left anterior descending artery issuitable for studying and practicing this method. The method is suitablefor therapeutic purposes in humans. This method of promoting tissueregeneration may be accomplished by stem cell engraftment. It isapplicable to the heart, and other organs, including the brain,pancreas, lung, liver, skin, and bone. SDF-1 can be delivered to thetissues by conventional local administration of a molecule describedherein or a composition comprising such molecule. SDF-1 can also bedelivered to the tissues by local administration of a cell comprising amolecule described herein or a composition comprising such molecule.Suitable cells include heart cells, brain cells, pancreatic cells, lungcells, liver cells, skin cells, bone cells, mesenchymal stem cells,progenitor cells, adult stem cells, and embryonic stem cells.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia and scimitarsyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of Fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's syndrome, trilogy of Fallot, and ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, scimitar syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve diseases include aortic valve insufficiency, aortic valvestenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau disease, Klippel-Trenaunay-Weber syndrome, Sturge-Webersyndrome, angioneurotic edema, aortic diseases, Takayasu's arteritis,aortitis, Leriche's syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, ataxia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subarachnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromboembolisms. Thromboses include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's syndrome, Churg-Strauss syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Additionally, ocular disorders associated with neovascularization whichcan be treated with the SDF-1 polynucleotides and polypeptides of thepresent invention (including SDF-1 agonists and SDF-1 antagonists)include, but are not limited to neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity, macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors, and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal., 85:704-710 (1978) and Gartner et al., Surv. Ophthal.,22:291-312 (1978).

Additionally, disorders which can be treated with the SDF-1polynucleotides and polypeptides of the present invention (includingSDF-1 agonists and SDF-1 antagonists) include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Polynucleotides and/or polypeptides of the invention, and/or agonistsand/or antagonists thereof, are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e.g., immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, small cellcarcinoma of the lung, stomach cancer, etc.), lymphoproliferativedisorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial,etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpes virusinfection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6;HHV-7, EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.),Helicobacter pylori infection, invasive Staphylococci, etc.), parasiticinfection, nephritis, bone disease (e.g., osteoporosis),atherosclerosis, pain, cardiovascular disorders (e.g.,neovascularization, hypovascularization or reduced circulation (e.g.,ischemic disease (e.g., myocardial infarction, stroke, etc.)), AIDS,allergy, inflammation, neurodegenerative disease (e.g., Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentaryretinitis, cerebellar degeneration, etc.), graft rejection (acute andchronic), graft vs. host disease, diseases due to osteomyelodysplasia(e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism,liver disease (e.g., acute and chronic hepatitis, liver injury, andcirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,diabetic nephropathy, diabetic neuropathy, diabetic retinopathy),influenza, asthma, psoriasis, glomerulonephritis, septic shock, andulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures).

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are also useful as an adjuvant to enhanceimmune responsiveness to specific antigen and/or anti-viral immuneresponses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) the immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or otherwise known in the art.

The uses of the SDF-1 polypeptides, include, but are not limited to, thetreatment or prevention of viral hepatitis, herpes viral infections,allergic reactions, adult respiratory distress syndrome, neoplasia,anaphylaxis, allergic asthma, allergen rhinitis, drug allergies (e.g.,to penicillin, cephalosporins), primary central nervous system lymphoma(PCNSL), glioblastoma, chronic lymphocytic leukemia (CLL),lymphadenopathy, autoimmune disease, graft versus host disease,rheumatoid arthritis, osteoarthritis, Graves' disease, acutelymphoblastic leukemia (ALL), lymphomas (Hodgkin's disease andnon-Hodgkin's lymphoma (NHL)), ophthalmopathy, uveoretinitis, theautoimmune phase of Type 1 diabetes, myasthenia gravis,glomerulonephritis, autoimmune hepatological disorder, autoimmuneinflammatory bowel disease, and Crohn's disease. In addition, the SDF-1polypeptides of the present invention may be employed to inhibitneoplasia, such as tumor cell growth. The combination of SDF-1 proteinwith immunotherapeutic agents such as IL-2 or IL-12 may result insynergistic or additive effects that would be useful for the treatmentof established cancers.

Antibodies

SDF-1-protein specific antibodies for use in the present invention canbe raised against the intact SDF-1 protein or an antigenic polypeptidefragment thereof. The protein or fragment may be presented with orwithout a carrier protein, such as an albumin, to an animal system (suchas rabbit or mouse); in general the polypeptide fragments aresufficiently immunogenic to produce a satisfactory immune responsewithout a carrier if they are at least about 25 amino acids in length.

Antibodies of the invention include polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, chimeric antibodies and, humanized antibodies, aswell as: hybrid (chimeric) antibody molecules (see, for example, Winteret al., Nature 349:293-299 (1991)); and U.S. Pat. No. 4,816,567);F(ab′)₂ and F(ab) fragments; Fv molecules (noncovalent heterodimers,see, for example, Inbar et al., Proc. Natl. Acad. Sci. 69:2659-2662(1972)); and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chainFv molecules (sFv) (see, e.g., Huston et al., Proc. Natl. Acad. Sci.85:5879-5883 (1980)); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al., Biochem. 31:1579-1584 (1992); Cumberet al., J. Immunology 149B: 120-126 (1992)); humanized antibodymolecules (see, e.g., Riechmann et al., Nature 332:323-327 (1988);Verhoeyan et al., Science 239:1534-1536 (1988)); and any functionalfragments obtained from such molecules, wherein such fragments retainspecific binding.

Methods of making monoclonal and polyclonal antibodies are known in theart. Monoclonal antibodies are generally antibodies having a homogeneousantibody population. The term is not limited regarding the species orsource of the antibody, nor is it intended to be limited by the mannerin which it is made. The term encompasses whole immunoglobulins.Polyclonal antibodies are generated by immunizing a suitable animal,such as a mouse, rat, rabbit, sheep or goat, with an antigen ofinterest, such as a stem cell transformed with a gene encoding anantigen. In order to enhance immunogenicity, the antigen can be linkedto a carrier prior to immunization. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., in order to enhance the immunogenicity thereof.

In addition, techniques developed for the production of chimericantibodies (Morrison et al., Proc. Natl. Acad. Sci., 81:851-855 (1984);Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature,314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used.Chimeric antibodies, i.e., antibodies in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, for example, humanized antibodies, andinsertion/deletions relating to cdr and framework regions, re suitablefor use in the invention.

The invention also includes humanized antibodies, i.e., those withmostly human immunoglobulin sequences. Humanized antibodies of theinvention generally refer to non-human immunoglobulins that have beenmodified to incorporate portions of human sequences. A humanizedantibody may include a human antibody that contains entirely humanimmunoglobulin sequences.

The antibodies of the invention may be prepared by any of a variety ofmethods. For example, cells expressing the SDF-1 protein or an antigenicfragment thereof can be administered to an animal in order to induce theproduction of sera containing polyclonal antibodies. A preparation ofSDF-1 protein can be prepared and purified to render it substantiallyfree of natural contaminants, and the preparation introduced into ananimal in order to produce polyclonal antisera with specific bindingactivity.

Antibodies of the invention specifically bind to their respectiveantigen(s); they may display high avidity and/or high affinity to aspecific polypeptide, or more accurately, to an epitope of an antigen.Antibodies of the invention may bind to one epitope, or to more than oneepitope. They may display different affinities and/or avidities todifferent epitopes on one or more molecules. When an antibody binds morestrongly to one epitope than to another, adjusting the bindingconditions can, in some instances, result in antibody binding almostexclusively to the specific epitope and not to any other epitopes on thesame polypeptide, and not to a polypeptide that does not comprise theepitope.

The invention also provides monoclonal antibodies and SDF-1 proteinbinding fragments thereof. Monoclonal antibodies of the invention can beprepared using hybridoma technology, for example, Kohler et al., Nature,256:495 (1975); Kohler et al., Eur. J. Immunol., 6:511 (1976); Kohleret. al., Eur. J. Immunol., 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp.563-681). In general, such procedures involve immunizing an animal(e.g., a mouse) with an SDF-1 protein antigen or with an SDF-1protein-expressing cell. Suitable cells can be recognized by theircapacity to bind anti-SDF-1 protein antibody. Such cells may be culturedin any suitable tissue culture medium; e.g., in Earle's modified Eagle'smedium supplemented with 10% fetal bovine serum (inactivated at about56° C.), and supplemented with about 10 grams/liter of nonessentialamino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml ofstreptomycin. The splenocytes of such mice are extracted and fused witha suitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; e.g., the parentmyeloma cell line (SP20), available from the American Type CultureCollection, Manassas, Va. After fusion, the resulting hybridoma cellsare selectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al., Gastroenterology, 80:225-232(1981).

SDF-1 AND SDF-1 Protein Antigen

Alternatively, antibodies capable of binding to the SDF-1 proteinantigen may be produced in a two-step procedure through the use ofanti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, SDF-1-protein specific antibodies are used to immunizean animal, e.g., a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theSDF-1 protein-specific antibody can be blocked by the SDF-1 proteinantigen. Such antibodies comprise anti-idiotypic antibodies to the SDF-1protein-specific antibody and can be used to immunize an animal toinduce formation of further SDF-1 protein-specific antibodies.

It will be appreciated that Fab and F(ab)₂ and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, SDF-1protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry. Humanizedchimeric monoclonal antibodies are suitable for in vivo use ofanti-SDF-1 in humans. Such humanized antibodies can be produced usinggenetic constructs derived from hybridoma cells producing the monoclonalantibodies described above. Methods for producing chimeric antibodiesare known in the art. See, for review, Morrison, Science, 229:1202(1985); Oi et al., BioTechniques, 4:214 (1986); Cabilly et al., U.S.Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature, 312:643 (1984); Neuberger et al., Nature,314:268 (1985).

Diagnosis

This invention is also related to the use of the genes of the presentinvention as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thenucleic acid sequences encoding the polypeptide of the presentinvention. Individuals carrying mutations in a gene of the presentinvention may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy, and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR, for example, as described by Saiki et al.,Nature, 324: 163-166 (1986), prior to analysis. RNA or cDNA may also beused for the same purpose. As an example, PCR primers complementary tothe nucleic acid encoding a polypeptide of the present invention can beused to identify and analyze mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled RNA or alternatively,radiolabeled antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetecting alterations in electrophoretic mobility of DNA fragments ingels run with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures, for example, asdescribed by Myers et al., Science, 230:1242 (1985).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method as shown in Cotton et al., Proc. Natl. Acad. Sci., USA,85:4397-4401 (1985). Thus, the detection of a specific DNA sequence maybe achieved by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes,(e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southernblotting of genomic DNA. In addition to more conventionalgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of SDF-1 proteins in various tissues. An over-expressionof these proteins compared to normal control tissue samples may detectthe presence of abnormal cellular proliferation, for example, a tumor.Assays used to detect protein levels in a host-derived sample arewell-known to those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays,“sandwich” assays, and other assays for the expression levels of thegenes encoding the SDF-1 proteins known in the art. Expression can beassayed by qualitatively or quantitatively measuring or estimating thelevel of SDF-1 protein, or the level of mRNA encoding SDF-1 protein, ina biological sample. Assays may be performed directly, for example, bydetermining or estimating absolute protein level or mRNA level, orrelatively, by comparing the SDF-1 protein or mRNA to a secondbiological sample. In performing these assays, the SDF-1 protein or mRNAlevel in the first biological sample is measured or estimated andcompared to a standard SDF-1 protein level or mRNA level; suitablestandards include second biological samples obtained from an individualnot having the disorder of interest. Standards may be obtained byaveraging levels of SDF-1 in a population of individuals not having adisorder related to SDF-1 expression. As will be appreciated in the art,once a standard SDF-1 protein level or mRNA level is known, it can beused repeatedly as a standard for comparison.

An ELISA assay, for example, as described by Coligan, et al., CurrentProtocols in Immunology, 1(2), Chap. 6, (1991), utilizes an antibodyprepared with specificity to a polypeptide antigen of the presentinvention. In addition, a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as a radioactive tag, a fluorescent tag, or an enzymatictag, e.g., a horseradish peroxidase. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein, e.g., bovineserum albumin. Next, the specific antibody, e.g., a monoclonal antibody,is incubated in the dish, during which time the antibody attaches to anypolypeptides of the present invention attached to the polystyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody, i.e., one linked to horseradish peroxidase is placed in thedish, resulting in the binding of the reporter antibody to any antibodybound to the protein of interest; unattached reporter antibody is thenremoved. Substrate, e.g., peroxidase, is then added to the dish, and theamount of signal produced color, e.g., developed in a given time periodprovides a measurement of the amount of a polypeptide of the presentinvention present in a given volume of patient sample when comparedagainst a standard.

A competition assay may be employed wherein antibodies specific to apolypeptide of the present invention are attached to a solid support,and labeled SDF-1, along with a sample derived from the host, are passedover the solid support. The label can be detected and quantified, forexample, by liquid scintillation chromatography, and the measurement canbe correlated to the quantity of the polypeptide of interest present inthe sample. A “sandwich” assay, similar to an ELISA assay, may beemployed, wherein a polypeptide of the present invention is passed overa solid support and binds to antibody modules attached to the solidsupport. A second antibody is then bound to the polypeptide of interest.A third antibody, which is labeled and specific to the second antibodyis then passed over the solid support and binds to the second antibody.The amount of antibody binding can be quantified; it correlates with theamount of the polypeptide of interest.

Biological samples of the invention can include any biological sampleobtained from a subject, body fluid, cell line, tissue culture, or othersource which contains SDF-1 protein or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovialfluid, and spinal fluid) which contain free SDF-1 protein, ovarian orrenal system tissue, and other tissue sources found to express completeor mature SDF-1 polypeptide or an SDF-1 receptor. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy mayprovide the source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem., 162:156-159 (1987). Levels ofmRNA encoding the SDF-1 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, PCR,reverse transcription in combination with PCR (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Assaying SDF-1 protein levels in a biological sample can be performedusing antibody-based techniques. For example, SDF-1 protein expressionin tissues can be studied with classical immunohistological methods, forexample, Jalkanen, M., et al., J. Cell. Biol., 101:976-985 (1985);Jalkanen, M., et al., J. Cell. Biol., 105:3087-3096 (1987). Otherantibody-based methods useful for detecting SDF-1 protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as glucoseoxidase, radioisotopes, and fluorescent labels, such as fluorescein andrhodamine, and biotin.

In addition to assaying SDF-1 protein levels in a biological sampleobtained from an individual, SDF-1 protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of SDF-1protein include those detectable by X-radiography, NMR, or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful to asubject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

An SDF-1 protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope, a radio-opaque substance, or a material detectable bynuclear magnetic resonance, is introduced, for example, parenterally,subcutaneously or intraperitoneally, into the subject to be examined foran immune system disorder. It will be understood in the art that thesize of the subject and the imaging system used will determine thequantity of imaging moiety needed to produce diagnostic images. Thelabeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain SDF-1 protein. In vivotumor imaging is described in Burchiel et al., ed., Chapter 13, TumorImaging: The Radiochemical Detection of Cancer, Masson Publishing Inc.(1982).

Formulations

The SDF-1 polypeptide compositions will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual subject, the site of delivery ofthe SDF-1 polypeptide composition, the method of administration, thescheduling of administration, and other factors known to practitioners.The effective amount of SDF-1 polypeptide for purposes herein is thusdetermined by such considerations.

The polypeptides, agonists, and antagonists of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition for parenteral administration.Such compositions comprise a therapeutically effective amount of thepolypeptide, agonist, or antagonist and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10micrograms/kg body weight and in most cases they will be administered inan amount not in excess of about 8 milligrams/kg body weight per day.

The polypeptides of the invention, and agonist and antagonist compoundswhich are polypeptides, may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo, i.e., genetherapy. Thus, for example, cells may be engineered with apolynucleotide (DNA or RNA) encoding for the polypeptide ex vivo; theengineered cells are then provided to a patient. Such methods arewell-known in the art. For example, cells may be engineered byprocedures known in the art by use of a retroviral particle containingRNA encoding for the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expressing thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a cell producing a retroviral particle containing RNAencoding the polypeptide of the present invention may be administered toa patient for the purpose of engineering cells in vivo and expressingthe polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by similar methods should beapparent to those skilled in the art from the teachings of the presentinvention. For example, the expression vehicle for engineering cells maybe other than a retroviral particle, for example, an adenovirus, whichmay be used to engineer cells in vivo after combination with a suitabledelivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia virus, spleen necrosis virus, retroviruses such as Rous sarcomavirus, Harvey sarcoma virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, myeloproliferativesarcoma virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney murine leukemia virus.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Vectors of theinvention include one or more promoters. Suitable promoters which may beemployed include, but are not limited to, the retroviral LTR; the SV40promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother homologous or heterologous promoter, for example, cellularpromoters such as eukaryotic cellular promoters including, but notlimited to, the histone, pol III, and β-actin promoters. Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, e.g., the adenoviral major late promoter;thymidine kinase (TK) promoters; and B19 parvovirus promoters.

Suitable promoters include the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAl promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs (including the modified retroviral LTRs hereinabovedescribed); the beta-actin promoter; and human growth hormone promoters.The promoter also may be the native promoter which controls the geneencoding the polypeptide. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

A retroviral plasmid vector can be employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, -2,-AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAm12, and DANcell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990).The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

SDF-1 “Knock-Outs” and Homologous Recombination

Endogenous gene expression can be reduced by inactivating or “knockingout” a gene of interest and/or its promoter using targeted homologousrecombination. (e.g., see Smithies et al., Nature, 317:230-234 (1985);Thomas & Capecchi, Cell, 51:503-512 (1987); Thompson et al., Cell,5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express, the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (see, e.g., Thomas & Capecchi(1987) supra; Thompson (1989), supra). However, this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Thecells are genetically engineered in vitro using recombinant DNAtechniques to introduce the coding sequence of polypeptides of theinvention into the cells, or alternatively, to disrupt the codingsequence and/or endogenous regulatory sequence associated with thepolypeptides of the invention, e.g., by transduction (using viralvectors, and/or vectors that integrate the transgene into the cellgenome) or transfection procedures, including, but not limited to, theuse of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes,etc. The coding sequence of the polypeptides of the invention can beplaced under the control of a strong constitutive or inducible promoteror promoter/enhancer to achieve expression, and secretion, of thepolypeptides of the invention. The engineered cells which express andsecrete the polypeptides of the invention can be introduced into thepatient systemically, e.g., in the circulation, or intraperitoneally.Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959, each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic Non-Human Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows, and non-human primates, e.g., baboons, monkeys, and chimpanzeesmay be used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce afounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pluripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, Intl. Rev. Cytol.115:171-229 (1989), which is incorporated by reference herein in itsentirety. Further, the contents of each of the documents recited in thisparagraph is herein incorporated by reference in its entirety. See also,U.S. Pat. No. 5,464,764 (Capecchi et al., Positive-Negative SelectionMethods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi et al., Cellsand Non-Human Organisms Containing Predetermined Genomic Modificationsand Positive-Negative Selection Methods and Vectors for Making Same);U.S. Pat. No. 4,736,866 (Leder et al., Transgenic Non-Human Animals);and U.S. Pat. No. 4,873,191 (Wagner et al., Genetic Transformation ofZygotes); each of which is hereby incorporated by reference in itsentirety. Any technique known in the art may be used to producetransgenic clones containing polynucleotides of the invention, forexample, nuclear transfer into enucleated oocytes of nuclei fromcultured embryonic, fetal, or adult cells induced to quiescence(Campbell et al., Nature 380:64-66 (1996); Wilmut et al., Nature385:810-813 (1997)), each of which is herein incorporated by referencein its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic or chimericanimals. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA 89:6232-6236(1992)). The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. It may be desired thatthe polynucleotide transgene be integrated into the chromosomal site ofthe endogenous gene, gene targeting is then suitable. Briefly, when sucha technique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous gene are designed for the purposeof integrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous gene. The transgene may also be selectively introduced into aparticular cell type, thus inactivating the endogenous gene in only thatcell type, by following, for example, the teaching of Gu et al. (Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited tooutbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knockout” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of SDF-1 polypeptides, studyingconditions and/or disorders associated with aberrant SDF-1 expression,and in screening for compounds effective in ameliorating such conditionsand/or disorders.

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, e.g., a purified antibody, in one or more containers. In aspecific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit. Thekits of the present invention may also comprise a control antibody whichdoes not react with the polypeptide of interest.

In another embodiment, the kits of the present invention comprise ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another embodiment of the present invention, the kit is a diagnostickit for use in screening serum containing antibodies specific againstproliferative and/or cancerous polynucleotides and polypeptides. Such akit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In embodiments, the kit may include arecombinantly produced or chemically synthesized polypeptide antigen.The polypeptide antigen of the kit may also be attached to a solidsupport.

In a further embodiment, the detecting means of the above-described kitincludes a solid support to which said polypeptide antigen is attached.Such a kit may also include a non-attached reporter-labeled anti-humanantibody. In this embodiment, binding of the antibody to the polypeptideantigen can be detected by binding of the said reporter-labeledantibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In an embodiment, the antibody is a monoclonalantibody. The detecting means of the kit may include a second, labeledmonoclonal antibody. Alternatively, or in addition, the detecting meansmay include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plates and/or filtermaterial. These attachment methods generally include non-specificadsorption of the protein to the support or covalent attachment of theprotein, typically through a free amine group, to a chemically reactivegroup on the solid support, such as an activated carboxyl, hydroxyl, oraldehyde group. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “an antigen”includes a mixture of two or more antigens, a reference to “a subjectpolypeptide” includes a plurality of such polypeptides, and reference to“the agent” includes reference to one or more agents and equivalentsthereof known to those skilled in the art, and so forth.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Moreover, it mustbe understood that the invention is not limited to the particularembodiments described, as such may, of course, vary. Further, theterminology used to describe particular embodiments is not intended tobe limiting, since the scope of the present invention will be limitedonly by its claim.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest the invention.

Further, all numbers expressing quantities of ingredients, reactionconditions, % purity, polypeptide and polynucleotide lengths, and soforth, used in the specification and claims, are modified by the term“about,” unless otherwise indicated. Accordingly, the numericalparameters set forth in the specification and claims are approximationsthat may vary depending upon the desired properties of the presentinvention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits, applying ordinary roundingtechniques.

Nonetheless, the numerical values set forth in the specific examples arereported as precisely as possible. Any numerical value, however,inherently contains certain errors from the standard deviation of itsexperimental measurement.

Patents and publications cited are incorporated by reference herein intheir entireties; references cited in such publications are alsoincorporated by reference in their entireties.

Tables

TABLE 1 SEQ ID NOS.: 1-23 FP ID SEQ.ID.NO.: (N1) SEQ.ID.NO.: (P1)SEQ.ID.NO.: (N0) Source ID HG1015480 SEQ.ID.NO.: 1 SEQ.ID.NO.: 9SEQ.ID.NO.: 17 CLN00235738_5pv1.a HG1015481 SEQ.ID.NO.: 2 SEQ.ID.NO.: 10SEQ.ID.NO.: 18 18093693 HG1015482 SEQ.ID.NO.: 3 SEQ.ID.NO.: 11SEQ.ID.NO.: 19 NP_000600:NM_000609 HG1015483 SEQ.ID.NO.: 4 SEQ.ID.NO.:12 SEQ.ID.NO.: 20 1220364:1220363 HG1015484 SEQ.ID.NO.: 5 SEQ.ID.NO.: 13SEQ.ID.NO.: 21 10334450:1220365 HG1015509 SEQ.ID.NO.: 6 SEQ.ID.NO.: 14CLN00235738_5pv1.a_exon2-4 HG1015510 SEQ.ID.NO.: 7 SEQ.ID.NO.: 15CLN00235738_5pv1.a_exon3-4 HG1015511 SEQ.ID.NO.: 8 SEQ.ID.NO.: 16CLN00235738_5pv1.a_exon4 HG1015522 SEQ.ID.NO.: 22CLN00235738_5pv1.a_3p_region HG1015525 SEQ.ID.NO.: 23CLN00235738_5pv1.a_5p_region

TABLE 2 ANNOTATION OF POLYPEPTIDE SEQUENCES Top Top Pred Human HumanProt Top Human Hit Hit % Match FP ID Source ID Len Accession ID TopHuman Hit Annot ID Length HG1015480 CLN00235738_5pv1.a 140gi|10334450|emb|CAC10202.1| bA20J15.1.2 (stromal cell-derived 100 92factor 1, isoform beta) [Homo sapiens] HG1015481 18093693 119gi|10334450|emb|CAC10202.1| bA20J15.1.2 (stromal cell-derived 100 92factor 1, isoform beta) [Homo sapiens] HG1015482 NP_000600:NM_000609 93gi|10834988|ref|NP_000600.1| chemokine (C—X—C motif) ligand 100 93 12(stromal cell-derived factor 1); stromal cell-derived factor 1 [Homosapiens] HG1015483 1220364: 89 gi|1220364|gb|AAB39333.1| pre-B cellstimulating factor 100 89 1220363 homologue HG1015484 10334450: 92gi|10334450|emb|CAC10202.1| bA20J15.1.2 (stromal cell-derived 100 921220365 factor 1, isoform beta) [Homo sapiens] HG1015509CLN00235738_5pv1.a_exon2-4 119 gi|10334450|emb|CAC10202.1| bA20J15.1.2(stromal cell-derived 56 92 factor 1, isoform beta) [Homo sapiens]HG1015510 CLN00235738_5pv1.a_exon3-4 80 no_human_hit HG1015511CLN00235738_5pv1.a_exon4 51 no_human_hit HG1015522CLN00235738_5pv1.a_3p_region 21 no_human_hit

TABLE 3 CHARACTERISTICS OF POLYPEPTIDE SEQUENCES Alternate Mature MaturePred Prot Tree Protein Signal Peptide Protein FP ID Source ID ClusterClass Len vote Coords Coords Coords TM Pfam HG1015480 CLN00235738_5pv1.a140 0.98 (22-140) (1-21) 0 IL8 HG1015481 18093693 119 0.98 (22-119)(1-21) 0 IL8 HG1015482 NP_000600: 180496 SECR 93 1 (22-93)  (1-21) 0 IL8NM_000609 HG1015483 1220364:1220363 180496 SECR 89 0.98 (22-89)  (1-21)0 IL8 HG1015484 10334450:1220365 180496 SECR 92 0.98 (22-92)  (1-21) 0IL8 HG1015509 CLN00235738_5pv1.a_exon2-4 119 0.21  (1-119) 0 IL8HG1015510 CLN00235738_5pv1.a_exon3-4 80 0.05 (1-80) 0 IL8 HG1015511CLN00235738_5pv1.a_exon4 51 0.08 (1-51) (27-51)  (12-26) 0 no_pfamHG1015522 CLN00235738_5pv1.a_3p_region 21 0.09 (1-21) (7-20) (21-21) 0no_pfam

TABLE 4 COMPARISON OF THE POLYPEPTIDES OF THE SEQUENCE LISTING WITHKNOWN POLYPEPTIDE SEQUENCES Length of Match % ID of FP ID vs Source PredProt between FP ID % ID of FP ID vs Source ID over Length of Source FPID Source ID Len and Source ID ID over Length of FP ID ID HG1015480CLN00235738_5pv1.a 140 — — — HG1015481 18093693 119 91 65% 76% HG1015482NP_000600: 93 88 63% 95% NM_000609 HG1015483 1220364:1220363 89 88 63%99% HG1015484 10334450:1220365 92 88 63% 96% HG1015509CLN00235738_5pv1.a_exon2-4 119 — — — HG1015510CLN00235738_5pv1.a_exon3-4 80 — — — HG1015511 CLN00235738_5pv1.a_exon451 — — — HG1015522 CLN00235738_5pv1.a_3p_region 21 — — —

TABLE 5 PFAM COORDINATES FP ID Source ID Pfam Coords. HG1015480CLN00235738_5pv1.a IL8 (22-87) HG1015481 18093693 IL8 (22-87) HG1015482NP_000600:NM_000609 IL8 (22-87) HG1015483 1220364:1220363 IL8 (22-87)HG1015484 10334450:1220365 IL8 (22-87) HG1015509CLN00235738_5pv1.a_exon2-4 IL8  (1-66) HG1015510CLN00235738_5pv1.a_exon3-4 IL8  (1-27) HG1015511CLN00235738_5pv1.a_exon4 no_pfam HG1015522 CLN00235738_5pv1.a_3p_regionno_pfam

1. A first isolated nucleic acid molecule comprising a firstpolynucleotide sequence chosen from (A) SEQ. ID. NOS.:1, 6-8, and 17;(B) a polynucleotide sequence encoding a polypeptide of SEQ. ID. NO.:9,14-16, 22-23; (C) biologically active fragments thereof; and (D)complements thereof. 2-4. (canceled)
 5. An isolated polypeptidecomprising an amino acid sequence, wherein the amino acid sequence ischosen from SEQ. ID. NO.:9, 14-16, and 22-23. 6-18. (canceled)
 19. Apolypeptide composition comprising the polypeptide of claim 5 and avehicle. 20-24. (canceled)
 25. A method of producing a recombinant hostcell comprising: (a) providing a composition comprising the nucleic acidmolecule of claim 1; (b) allowing a host cell to come into contact withthe nucleic acid to form a recombinant host cell.
 26. (canceled)
 27. Amethod of producing a polypeptide comprising: (a) providing the nucleicacid of claim 1; and (b) expressing the nucleic acid molecule in anexpression system to produce the polypeptide. 28-30. (canceled)
 31. Adiagnostic kit comprising an antibody that specifically binds to thepolypeptide of claim 5 or a biologically active fragment thereof. 32.(canceled)
 33. A method of determining the presence of the nucleic acidmolecule of claim 1 in a sample comprising: (a) providing the nucleicacid molecule of claim 1; (b) allowing the nucleic acid molecule ofclaim 1 to interact with the sample under conditions that allow forspecific binding; and (c) determining whether specific binding hasoccurred.
 34. A method of determining the presence of an antibodyspecific to the polypeptide of claim 5 in a sample comprising: (a)providing a composition comprising the polypeptide of claim 5; (b)allowing the polypeptide to interact with the sample under conditionsthat allow for specific binding; and (c) determining whether specificbinding has occurred between the polypeptide and the antibody.
 35. Anantibody that specifically binds to or interferes with an activity ofthe polypeptide of claim
 5. 36-38. (canceled)
 39. A method of treating aB-cell deficiency in a subject comprising: (a) providing a compositioncontaining a polypeptide of claim 5 and a vehicle; and (b) administeringthe composition to the subject. 40-45. (canceled)
 46. A method oftreatment of a platelet deficiency in a subject comprising: (a)providing a composition containing a polypeptide of claim 5 and avehicle; and (b) administering the composition to the subject. 47-49.(canceled)
 50. A method of stimulating lymphocyte growth orproliferation in a subject comprising: (a) providing a compositioncontaining a polypeptide of claim 5 and a vehicle; and (b) administeringthe composition to the subject. 51-53. (canceled)
 54. A method fortreating an adverse effect of a cancer therapy in a subject comprising:(a) administering a composition containing a polypeptide of claim 5 tothe subject; (b) collecting a population of stem cells from the subject;and (c) administering the population of stem cells to the subject.55-60. (canceled)
 61. A method of treating diabetes in a subjectcomprising: (a) providing a composition containing a polypeptide ofclaim 5 and a vehicle; and (b) administering the composition to thesubject. 62-63. (canceled)
 64. A method of promoting angiogenesis in asubject comprising: (a) providing a composition containing a polypeptideof claim 5 and a vehicle; and (b) administering the composition to thesubject. 65-66. (canceled)
 67. A method of modulating an immune responsein a subject comprising: (a) providing a modulator of a polypeptide ofclaim 5; and (b) administering the modulator to the subject. 68-74.(canceled)
 75. A method for treating or preventing an infection in asubject comprising: (a) providing a composition containing a polypeptideof claim 5 and a vehicle; and (b) administering the composition to thesubject. 76-79. (canceled)
 80. A method for treating or preventing anischemic disease in a subject comprising: (a) providing a compositioncontaining a polypeptide of claim 5 and a vehicle; and (b) administeringthe composition to the subject. 81-83. (canceled)
 84. A method ofinhibiting tumor growth in a subject comprising: (a) providing acomposition containing a polypeptide of claim 5 and a vehicle; and (b)administering the composition to the subject. 85-87. (canceled)
 88. Amethod of treating a cancer in a subject comprising: (a) providing acomposition containing a polypeptide of claim 5 and a vehicle; and (b)administering the composition to the subject. 89-92. (canceled)
 93. Amethod of treating an allergy in a subject comprising: (a) providing acomposition containing a polypeptide of claim 5 and a vehicle; and (b)administering the composition to the subject. 94-100. (canceled)
 101. Amethod of modulating an immune condition in a subject, comprising: (a)providing a modulator of a polypeptide of claim 5; and (b) administeringthe modulator to the subject. 102-106. (canceled)
 107. A method ofenhancing an immune response to a vaccine in a subject comprising: (a)providing a polypeptide composition comprising a substantially purifiedpolypeptide of claim 5; (b) providing a vaccine composition; and (c)administering the polypeptide composition and the vaccine composition tothe subject. 108-120. (canceled)
 121. A method for promoting tissueregeneration in a subject comprising: (a) providing a polypeptidecomprising an amino acid sequence chosen from SEQ. ID. NOS.:9 and 14-16;and (b) administering the polypeptide to the subject. 122-131.(canceled)
 132. A cell transfected with the nucleic acid molecule ofclaim
 1. 133. (canceled)
 134. A method of treating a disease in asubject who can benefit from receiving the polypeptide of claim 5,wherein the disease is selected from a cardiovascular disease, braindisease, bone disease, lung disease, liver disease, skin disease, burn,stroke, trauma, and an injury, comprising: (a) providing a compositioncomprising the polypeptide of claim 5; and (b) administering thecomposition to the subject. 135-146. (canceled)
 147. A polypeptidecomprising the amino acid sequence of amino acids 22-88 ofCLN00235738_(—)5pv1.a and further comprising at least one additionalamino acid chosen from amino acids 89-140, wherein polypeptide comprisesa contiguous sequence of amino acids chosen from CLN00235738_(—)5pv1.aas shown in FIG. 1.